Antisense antiviral compound and method for treating ssRNA viral infection

ABSTRACT

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridae and Hepeviridae families in the treatment of a viral infection. The antisense antiviral compounds are substantially uncharged morpholino oligonucleotides having a sequence of 12-40 subunits, including at least 12 subunits having a targeting sequence that is complementary to a region associated with stem-loop secondary structure within the 5′-terminal end 40 bases of the positive-sense RNA strand of the virus.

This application claims priority to U.S. provisional patent applicationNo. 60/611,063 filed on Sep. 16, 2004, which is incorporated herein inits entirety by reference.

FIELD OF THE INVENTION

This invention relates to antisense oligonucleotide compounds for use intreating a Flavivirus, Hepacivirus, Enterovirus, Rhinovirus,Hepatovirus, Apthovirus, Hepevirus, Coronavirus, Arterivirus, Vesivirus,Norovirus, Mamastrovirus, Alphavirus, and Rubivirus infection andantiviral treatment methods employing the compounds.

REFERENCES

The following references are related to the background or the invention.

-   Banerjee, R. and A. Dasgupta (2001). “Interaction of picornavirus 2C    polypeptide with the viral negative-strand RNA.” J Gen Virol 82(Pt    11): 2621-7.-   Banerjee, R. and A. Dasgupta (2001). “Specific interaction of    hepatitis C virus protease/helicase NS3 with the 3′-terminal    sequences of viral positive- and negative-strand RNA.” J Virol    75(4): 1708-21.-   Banerjee, R., A. Echeverri, et al. (1997). “Poliovirus-encoded 2C    polypeptide specifically binds to the 3′-terminal sequences of viral    negative-strand RNA.” J Virol 71(12): 9570-8.-   Banerjee, R., W. Tsai, et al. (2001). “Interaction of    poliovirus-encoded 2C/2BC polypeptides with the 3′ terminus    negative-strand cloverleaf requires an intact stem-loop b.” Virology    280(1): 41-51.-   Blommers, M. J., U. Pieles, et al. (1994). “An approach to the    structure determination of nucleic acid analogues hybridized to RNA.    NMR studies of a duplex between 2′-OMe RNA and an oligonucleotide    containing a single amide backbone modification.” Nucleic Acids Res    22(20): 4187-94.-   Gait, M. J., A. S. Jones, et al. (1974). “Synthetic-analogues of    polynucleotides XII. Synthesis of thymidine derivatives containing    an oxyacetamido- or an oxyformamido-linkage instead of a    phosphodiester group.” J Chem Soc [Perkin 1] 0(14): 1684-6.-   Holland, J. (1993). Emerging Virus. S. S. Morse. New York and    Oxford, Oxford University Press: 203-218.-   Lesnikowski, Z. J., M. Jaworska, et al. (1990). “Octa(thymidine    methanephosphonates) of partially defined stereochemistry: synthesis    and effect of chirality at phosphorus on binding to    pentadecadeoxyriboadenylic acid.” Nucleic Acids Res 18(8): 2109-15.-   Markoff, L. (2003). “5′- and 3′-noncoding regions in flavivirus    RNA.” Adv Virus Res 59: 177-228.-   Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate    analogs of dinucleosides. 3′-Thymidinyl 5′-thymidinyl carbonate,    3′-thymidinyl 5′-(5-fluoro-2′-deoxyuridinyl) carbonate, and    3′-(5-fluoro-2′-deoxyuridinyl) 5′-thymidinyl carbonate.” J Med Chem    12(1): 154-7.-   Moulton, H. M., M. H. Nelson, et al. (2004). “Cellular uptake of    antisense morpholino oligomers conjugated to arginine-rich    peptides.” Bioconjug Chem 15(2): 290-9.-   Murray, R. and e. al. (1998). Medical Microbiology. St. Louis, Mo.,    Mosby Press: 542-543.-   Neuman, B. W., D. A. Stein, et al. (2004). “Antisense    Morpholino-Oligomers Directed against the 5′ End of the Genome    Inhibit Coronavirus Proliferation and Growth{dagger}.” J. Virol.    78(11): 5891-5899.-   O'Ryan, M. (1992). Clinical Virology Manual. S. Spector and G.    Lancz. New York, Elsevier Science: 361-196.-   Pardigon, N., E. Lenches, et al. (1993). “Multiple binding sites for    cellular proteins in the 3′ end of Sindbis alphavirus minus-sense    RNA.” J Virol 67(8): 5003-11.-   Pardigon, N. and J. H. Strauss (1992). “Cellular proteins bind to    the 3′ end of Sindbis virus minus-strand RNA.” J Virol 66(2):    1007-15.-   Paul, A. V. (2002). Possible unifying mechanism of picornavirus    genome replication. Molecular Biology of Picornaviruses. B. L.    Semler and E. Wimmer. Washington, D.C., ASM Press: 227-246.-   Roehl, H. H., T. B. Parsley, et al. (1997). “Processing of a    cellular polypeptide by 3CD proteinase is required for poliovirus    ribonucleoprotein complex formation.” J Virol 71(1): 578-85.-   Roehl, H. H. and B. L. Semler (1995). “Poliovirus infection enhances    the formation of two ribonucleoprotein complexes at the 3′ end of    viral negative-strand RNA.” J Virol 69(5): 2954-61.-   Smith, A. W., D. E. Skilling, et al. (1998). “Calicivirus emergence    from ocean reservoirs: zoonotic and interspecies movements.” Emerg    Infect Dis 4(1): 13-20.-   Summerton, J. and D. Weller (1997). “Morpholino antisense oligomers:    design, preparation, and properties.” Antisense Nucleic Acid Drug    Dev 7(3): 187-95.-   Xu, W. Y. (1991). “Viral haemorrhagic disease of rabbits in the    People's Republic of China: epidemiology and virus    characterisation.” Rev Sci Tech 10(2): 393-408.-   Zuker, M. (2003). “Mfold web server for nucleic acid folding and    hybridization prediction.” Nucleic Acids Res 31(13): 3406-15.

BACKGROUND OF THE INVENTION

Single-stranded RNA (ssRNA) viruses cause many diseases in wildlife,domestic animals and humans. These viruses are genetically andantigenically diverse, exhibiting broad tissue tropisms and a widepathogenic potential. The incubation periods of some of the mostpathogenic viruses, e.g. the caliciviruses, are very short. Viralreplication and expression of virulence factors may overwhelm earlydefense mechanisms (Xu 1991) and cause acute and severe symptoms.

There are no specific treatment regimes for many viral infections. Theinfection may be serotype specific and natural immunity is often briefor absent (Murray and al. 1998). Immunization against these virulentviruses is impractical because of the diverse serotypes. RNA virusreplicative processes lack effective genetic repair mechanisms, andcurrent estimates of RNA virus replicative error rates are such thateach genomic replication can be expected to produce one to ten errors,thus generating a high number of variants (Holland 1993). Often, theserotypes show no cross protection such that infection with any oneserotype does not protect against infection with another. For example,vaccines against the vesivirus genus of the caliciviruses would have toprovide protection against over 40 different neutralizing serotypes(Smith, Skilling et al. 1998) and vaccines for the other genera of theCaliciviridae are expected to have the same limitations.

Thus, there remains a need for an effective antiviral therapy in severalvirus families, including small, single-stranded, positive-sense RNAviruses in the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae,Arteriviridae, Coronaviridae, Astroviridae or Hepeviridae families.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a method of producing ananti-viral compound effective in inhibiting replication within a hostcell of an RNA virus having a single-stranded, positive-sense genome andselected from one of the Flaviviridae, Picornoviridae, Caliciviridae,Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridaefamilies. The method includes first identifying as a viral targetsequence, a region within the 5′-terminal 40 bases of the positivestrand of the infecting virus whose sequence is capable of forminginternal stem-loop secondary structure. There is then constructed, bystep-wise solid-phase synthesis, an oligonucleotide analog compoundcharacterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases, and

(iv) having a targeting sequence of at least 12 subunits that iscomplementary to the virus-genome region capable of forming internalduplex structure, and

(v) an ability to form with the viral target sequence, a heteroduplexstructure (i) composed of the positive sense strand of the virus and theoligonucleotide compound, and (ii) characterized by a Tm of dissociationof at least 45° C. and disruption of such stem-loop structure.

The target sequence may be identified by obtaining analyzing the5′-terminal sequences, e.g., the 5′-terminal 40 bases by a computerprogram capable of performing secondary structure predictions based on asearch for the minimal free energy state of the input RNA sequence.

The invention includes, in another aspect, a method of inhibiting in amammalian host cell, replication of an infecting RNA virus having asingle-stranded, positive-sense genome and selected from one of theFlaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae,Coronaviridae, Astroviridae or Hepeviridae families. The method includesadministering to the infected host cells, a virus-inhibitory amount ofan oligonucleotide analog compound characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases, and

(iv) having a targeting sequence of at least 12 subunits that iscomplementary to a region within the 5′-terminal 40 bases of thepositive-strand viral genome that is capable of forming internalstem-loop secondary structure. The compound is effective, whenadministered to the host cells, to form a heteroduplex structure (i)composed of the positive sense strand of the virus and theoligonucleotide compound, and (ii) characterized by a Tm of dissociationof at least 45° C. and disruption of such stem-loop secondary structure.The compound may be administered to a mammalian subject infected withthe virus, or at risk of infection with the virus.

The compound may be composed of morpholino subunits linked by uncharged,phosphorus-containing intersubunit linkages, joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.In one embodiment, the intersubunit linkages are phosphorodiamidatelinkages, such as those having the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino,e.g., wherein X=NR₂, where each R is independently hydrogen or methyl.

The compound may be a covalent conjugate of an oligonucleotide analogmoiety capable of forming such a heteroduplex structure with thepositive sense strand of the virus, and an arginine-rich polypeptideeffective to enhance the uptake of the compound into host cells.

In a related aspect, the invention includes a heteroduplex complexformed between:

(a) a region within the 5′-terminal 40 bases of the positive strand RNAof an RNA virus having a single-stranded, positive-sense RNA genome andselected from one of the Flaviviridae, Picornoviridae, Caliciviridae,Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridaefamilies, which region is capable of forming internal stem-loopsecondary structure, and

(b) an oligonucleotide analog compound characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits that iscomplementary to a region associated with such stem-loop secondarystructure within the 5′-terminal end 40 bases of the positive-sense RNAstrand of the virus,

where said heteroduplex complex has a Tm of dissociation of at least 45°C. and disruption of such stem-loop secondary structure.

An exemplary compound is composed of morpholino subunits linked byuncharged, phosphorus-containing intersubunit linkages, joining amorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit. The compound may have phosphorodiamidate linkages,such as in the structure

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In apreferred compound, X=NR₂, where each R is independently hydrogen ormethyl. The compound may be the oligonucleotide analog alone or aconjugate of the analog and an arginine-rich polypeptide capable ofenhancing the uptake of the compound into host cells.

The invention is also directed to a method for detecting the presence ofa viral infection by an RNA virus having a single-stranded,positive-sense RNA genome and selected from one of the Flaviviridae,Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae,Coronaviridae, Astroviridae or Hepeviridae families a in a mammaliansubject, or for confirming the presence of an effective interactionbetween such a virus infecting a mammalian subject and an antisenseoligonucleotide analog compound directed against the virus. Inpracticing the method, the subject is administered an oligonucleotideanalog compound having (a) a sequence of 12-40 subunits, including atargeting sequence of at least 12 subunits that is complementary to aregion associated with stem-loop secondary structure within the5′-terminal end 40 bases of the positive-sense RNA strand of the virus,(b) morpholino subunits linked by uncharged, phosphorus-containingintersubunit linkages, each linkage joining a morpholino nitrogen of onesubunit to a 5′ exocyclic carbon of an adjacent subunit, and (c) capableof forming with the positive-strand viral ssRNA genome, a heteroduplexstructure characterized by a Tm of dissociation of at least 45° C. anddisruption of the stem-loop secondary structure.

At a selected time after the compound is administered, a sample of abody fluid is obtained from the subject, and assayed for the presence ofa nuclease-resistant heteroduplex comprising the antisenseoligonucleotide complexed with a complementary-sequence 5′-end region ofthe positive-strand RNA of the virus.

In still another aspect, the invention includes an oligonucleotideanalog compound for use in inhibiting replication in mammalian hostcells of an RNA virus having a single-stranded, positive-sense RNAgenome and selected from the Flaviviridae, Picornoviridae,Caliciviridae, Togaviridae, or Coronaviridae families and hepatitis Evirus. The compound is characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits that iscomplementary to a region associated with stem-loop secondary structurewithin the 5′-terminal end 40 bases of the positive-sense RNA strand ofthe virus, and

(v) capable of forming with the positive-strand viral ssRNA genome, aheteroduplex structure having a Tm of dissociation of at least 45° C.and disruption of such stem-loop secondary structure.

An exemplary compound is composed of morpholino subunits linked byuncharged, phosphorus-containing intersubunit linkages, joining amorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit. The compound may have phosphorodiamidate linkages,such as in the structure

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In apreferred compound, X=NR₂, where each R is independently hydrogen ormethyl. The compound may be the oligonucleotide analog alone or aconjugate of the analog and an arginine-rich polypeptide capable ofenhancing the uptake of the compound into host cells.

For treatment of a Flavivirus or Hepacivirus as given below, thetargeting sequence is complementary to a region associated withstem-loop secondary structure within one of the following sequences:

(i) SEQ ID NO. 1, for St Louis encephalitis virus;

(ii) SEQ ID NO. 2, for Japanese encephalitis virus;

(iii) SEQ ID NO. 3, for a Murray Valley encephalitis virus;

(iv) SEQ ID NO. 4, for a West Nile fever virus;

(v) SEQ ID NO. 5, for a Yellow fever virus

(vi) SEQ ID NO. 6, for a Dengue Type-2 virus;

(vii) SEQ ID NO. 7, for a Hepatitis C virus;

(viii) SEQ ID NO. 8, for a tick-borne encephalitis virus;

(ix) SEQ ID NO. 9, for Omsk hemorrhagic fever virus; and

(x) SEQ ID NO. 10, for Powassan virus.

Exemplary targeting sequences for these viruses include the followingsequences, or portions of these sequences that overlap with one or moreregions of duplex secondary structure in the associated target sequence:

(i) SEQ ID NOS. 41 and 42, for St Louis encephalitis virus;

(ii) SEQ ID NOS. 43 and 44, for Japanese encephalitis virus;

(iii) SEQ ID NOS. 45 and 46, for a Murray Valley encephalitis virus;

(iv) SEQ ID NOS. 47 and 48, for a West Nile fever virus;

(v) SEQ ID NOS. 49 and 50, for a Yellow fever virus

(vi) SEQ ID NOS. 51, 52, for a Dengue virus;

(vii) SEQ ID NOS. 53 and 54, for a Hepatitis C virus;

(viii) SEQ ID NOS. 55 and 56, for a tick-borne encephalitis virus;

(ix) SEQ ID NOS. 57 and 58, for Omsk hemorrhagic fever virus; and

(x) SEQ ID NOS. 59 and 60, for Powassan virus.

For treatment of an Enterovirus, Rhinovirus, Hepatovirus or Aphthovirusthe targeting sequence is complementary to a region associated withstem-loop secondary structure within one of the following sequences:

(i) SEQ ID NO. 11, for a polio virus of the Mahoney and Sabin strains;

(ii) SEQ ID NO. 12, for a Human enterovirus A;

(iii) SEQ ID NO. 13, for a Human enterovirus B;

(iv) SEQ ID NO. 14, for a Human enterovirus C;

(v) SEQ ID NO. 15, for a Human enterovirus D;

(vi) SEQ ID NO. 16, for a Human enterovirus E;

(vii) SEQ ID NO. 17, for a Bovine enterovirus;

(viii) SEQ ID NO. 18, for Human rhinovirus 89;

(ix) SEQ ID NO. 19, for Human rhinovirus B;

(x) SEQ ID NO. 20, for Foot-and-mouth disease virus; and

(xi) SEQ ID NO. 21, for a hepatitis A virus.

Exemplary targeting sequences for these viruses include the followingsequences, or portions of these sequences that overlap with one or moreregions of duplex secondary structure in the associated target sequence:

(i) SEQ ID NOS. 61 and 62, for a polio virus of the Mahoney and Sabinstrains;

(ii) SEQ ID NOS. 63 and 64, for a Human enterovirus A;

(iii) SEQ ID NOS. 65 and 66, for a Human enterovirus B;

(iv) SEQ ID NOS. 67 and 68, for a Human enterovirus C;

(v) SEQ ID NOS. 69 and 70, for a Human enterovirus D;

(vi) SEQ ID NOS. 71 and 72, for a Human enterovirus E;

(vii) SEQ ID NOS. 73 and 74, for a Bovine enterovirus;

(viii) SEQ ID NOS. 75 and 76, for Human rhinovirus 89;

(ix) SEQ ID NOS. 77 and 78, for Human rhinovirus B;

(x) SEQ ID NOS. 79 and 80, for Foot-and-mouth disease virus; and

(xi) SEQ ID NOS. 81 and 82, for a hepatitis A virus.

For treatment of a Calicivirus or Norovirus the targeting sequence iscomplementary to a region associated with stem-loop secondary structurewithin one of the following sequences:

(i) SEQ ID NO. 22, for a Feline Calicivirus;

(ii) SEQ ID NO. 23, for a Canine Calicivirus;

(iii) SEQ ID NO. 24, for a Porcine enteric calicivirus;

(iv) SEQ ID NO. 25, for Calicivirus strain NB; and

(v) SEQ ID NO. 26, for a Norwalk virus.

Exemplary targeting sequences for these viruses include the followingsequences, or portions of these sequences that overlap with one or moreregions of duplex secondary structure in the associated target sequence:

(i) SEQ ID NOS. 83 and 84, for a Feline Calicivirus;

(ii) SEQ ID NOS. 85 and 86, for a Canine Calicivirus;

(iii) SEQ ID NOS. 87 and 88, for a Porcine enteric calicivirus;

(iv) SEQ ID NOS. 89 and 90, for Calicivirus strain NB; and

(v) SEQ ID NOS. 91 and 92, for a Norwalk virus.

For treatment of the Hepevirus, Hepatitis E virus, the targetingsequence is complementary to a region associated with stem-loopsecondary structure within the sequence identified as SEQ ID NO: 27.Exemplary targeting sequences include SEQ ID NOS: 93 and 94, or portionsthereof that overlap with one or more regions of secondary structure inthe associated target sequence.

For treatment of a Rubivirus or Alphavirus the targeting sequence iscomplementary to a region associated with stem-loop secondary structurewithin one of the following sequences:

(i) SEQ ID NO. 28, for Rubella virus;

(ii) SEQ ID NO. 38, for Eastern equine encephalitis virus;

(iii) SEQ ID NO. 39, for Western equine encephalitis virus; and

(iv) SEQ ID NO. 40, for Venezuelan equine encephalitis virus.

Exemplary targeting sequences for each of these viruses are identifiedby the following sequence ID numbers, or portions of these sequencesthat overlap with one or more regions of duplex secondary structure inthe associated target sequence:

(i) SEQ ID NOS. 95 and 96, for Rubella virus;

(ii) SEQ ID NOS. 115 and 116, for Eastern equine encephalitis virus;

(iii) SEQ ID NOS. 117 and 118, for Western equine encephalitis virus;and

(iv) SEQ ID NOS. 119 and 120, for Venezuelan equine encephalitis virus

For treatment of a Coronavirus or Arterivirus the targeting sequence iscomplementary to a region associated with stem-loop secondary structurewithin one of the following sequences:

(i) SEQ ID NO. 29, for SARS coronavirus TOR2;

(ii) SEQ ID NO. 30, for Porcine epidemic diarrhea virus;

(iii) SEQ ID NO. 31, for Transmissible gastroenteritis virus;

(iv) SEQ ID NO. 32, for Bovine coronavirus;

(v) SEQ ID NO. 33, for Human coronavirus 229E;

(vi) SEQ ID NO. 34, for Murine hepatitis virus; and

(vii) SEQ ID NO. 35, for Porcine reproductive and respiratory syndromevirus.

Exemplary targeting sequences for each of these viruses are identifiedby the following sequence ID numbers, or portions of these sequencesthat overlap with one or more regions of duplex secondary structure inthe associated target sequence:

(i) SEQ ID NOS. 97 and 98, for SARS coronavirus TOR2;

(ii) SEQ ID NOS. 99 and 100, for Porcine epidemic diarrhea virus;

(iii) SEQ ID NOS. 101 and 102, for Transmissible gastroenteritis virus;

(iv) SEQ ID NOS. 103 and 104, for Bovine coronavirus;

(v) SEQ ID NOS. 105 and 106, for Human coronavirus 229E;

(vi) SEQ ID NOS. 107 and 108, for Murine hepatitis virus; and

(vii) SEQ ID NOS. 109 and 110, for Porcine reproductive and respiratorysyndrome virus.

For treatment of a Mamastrovirus, Human astrovirus, the targetingsequence is complementary to a region associated with stem-loopsecondary structure within the sequence identified as SEQ ID NO: 37.Exemplary targeting sequences are SEQ ID NOS. 113 and 114, or portionsof these sequences that overlap with one or more regions of duplexsecondary structure in the associated target sequence.

For treatment of an Equine arteritis virus, the targeting sequence iscomplementary to a region associated with stem-loop secondary structurewithin the sequence identified as SEQ ID NO: 36. Exemplary targetingsequences are SEQ ID NOS. 111, 112, or portions of these sequences thatoverlap with one or more regions of duplex secondary structure in theassociated target sequence.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show the backbone structures of various oligonucleotideanalogs with uncharged backbones;

FIGS. 2A-2D show the repeating subunit segment of exemplary morpholinooligonucleotides;

FIGS. 3A-3E are schematic diagrams of genomes of exemplary viruses andviral target sites;

FIGS. 4A-4E show examples of predicted secondary structures of 5′ endterminal positive-strand regions for exemplary viruses; FIG. 4A—JEV, SEQID NO.:2; FIG. 4A—MVEV, SEQ ID NO.:3; FIG. 4A—WNV, SEQ ID NO.:4; FIG.4A—YFV, SEQ ID NO.:5; FIG. 4A—DEN2, SEQ ID NO.:6; FIG. 4A—HCV, SEQ IDNO.:7; FIG. 4B—PV, SEQ ID NO.:11; FIG. 4B—HEV-A, SEQ ID NO.:12; FIG.4B—HEV-B, SEQ ID NO.:13; FIG. 4B—HEV-C, SEQ ID NO.:14; FIG. 4B—HEV-E,SEQ ID NO.:16; FIG. 4B—BEV, SEQ ID NO.:17; FIG. 4B—HRV-89, SEQ IDNO.:18; FIG. 4B—HRV-B, SEQ ID NO.:19; FIG. 4B—FMDV, SEQ ID NO.:20; FIG.4B—HAV, SEQ ID NO.:21; FIG. 4C—TBEV, SEQ ID NO.:8; FIG. 4C—OHFV, SEQ IDNO.:9; FIG. 4C—Powassan, SEQ ID NO.:10; FIG. 4C—FCV, SEQ ID NO.:22; FIG.4C—CaCV, SEQ ID NO.:23; FIG. 4C—PoCV, SEQ ID NO.:24; FIG. 4C—NV, SEQ IDNO.:26; FIG. 4C—RUBV, SEQ ID NO.:28; FIG. 4D—CVNB, SEQ ID NO.:25; FIG.4D—HEV, SEQ ID NO.:27; FIG. 4D—SARS, SEQ ID NO.:29; FIG. 4D—PEDV, SEQ IDNO.:30; FIG. 4D—TGEV, SEQ ID NO.:31; FIG. 4D—BCoV, SEQ ID NO.:32; FIG.4D—HCoV-229E, SEQ ID NO.:33; FIG. 4D—MHV, SEQ ID NO.:34; FIG. 4E—PRRSV,SEQ ID NO.:35; FIG. 4E—HAstV, SEQ ID NO.:37; FIG. 4E—EEEV, SEQ IDNO.:38; FIG. 4E—WEEV, SEQ ID NO.:39; FIG. 4E—VEEV, SEQ ID NO.:40; and

FIGS. 5A-5D show the inhibition of Dengue virus replication in infectedVero cells in the presence of an antisense oligomer that targets the 5′positive-strand terminal region of Dengue virus types 1-4.

FIG. 6 shows that PMO can reduce PRRSV replication as measured by viraltiter.

FIG. 7 shows in vitro PMO treatment of CRL11171 cells inoculated withPRRSV have reduced cell pathogenic effects.

FIG. 8 shows the reduction of TBEV replication in vitro in the presencePMO targeting the 5′ terminal region of TBEV.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

The terms “oligonucleotide analog” refers to an oligonucleotide having(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and (ii) optionally, modified sugar moieties, e.g.,morpholino moieties rather than ribose or deoxyribose moieties. Theanalog supports bases capable of hydrogen bonding by Watson-Crick basepairing to standard polynucleotide bases, where the analog backbonepresents the bases in a manner to permit such hydrogen bonding in asequence-specific fashion between the oligonucleotide analog moleculeand bases in a standard polynucleotide (e.g., single-stranded RNA orsingle-stranded DNA). Preferred analogs are those having a substantiallyuncharged, phosphorus containing backbone.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 60-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 8 and 40subunits, typically about 8-25 subunits, and preferably about 12 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligonucleotide analog” is an oligonucleotide analogcomposed of morpholino subunit structures of the form shown in FIGS.2A-2D, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil orthymine. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337,all of which are incorporated herein by reference.

The subunit and linkage shown in FIG. 2B are used for six-atomrepeating-unit backbones, as shown in FIG. 3B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 2B,where X=NH₂, NHR, or NR₂ (where R is lower alkyl, preferably methyl),Y=O, and Z=O, and P_(i) and P_(j) are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide. Also preferred are structures having an alternatephosphorodiamidate linkage, where, in FIG. 2B, X=lower alkoxy, such asmethoxy or ethoxy, Y=NH or NR, where R is lower alkyl, and Z=O.

The term “substituted”, particularly with respect to an alkyl, alkoxy,thioalkoxy, or alkylamino group, refers to replacement of a hydrogenatom on carbon with a heteroatom-containing substituent, such as, forexample, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino,imino, oxo (keto), nitro, cyano, or various acids or esters such ascarboxylic, sulfonic, or phosphonic. It may also refer to replacement ofa hydrogen atom on a heteroatom (such as an amine hydrogen) with analkyl, carbonyl or other carbon containing group.

As used herein, the term “target”, relative to the viral genomic RNA,refers to a viral genomic RNA, and specifically, to a region associatedwith stem-loop secondary structure within the 5′-terminal end 40 basesof the positive-sense RNA strand of a single-stranded RNA (ssRNA) virusdescribed herein.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide analog is directed, that is, the sequence towhich the oligonucleotide analog will hybridize by Watson-Crick basepairing of a complementary sequence. As will be seen, the targetsequence may be a contiguous region of the viral positive-strand RNA, ormay be composed of complementary fragments of both the 5′ and 3′sequences involved in secondary structure.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in an analog having 20 bases, only12-14 may be targeting sequences. Typically, the targeting sequence isformed of contiguous bases in the analog, but may alternatively beformed of non-contiguous sequences that when placed together, e.g., fromopposite ends of the analog, constitute sequence that spans the targetsequence. As will be seen, the target and targeting sequences areselected such that binding of the analog to a region within the5′-terminal end 40 bases of the positive-sense RNA strand of the virusacts to disrupt secondary structure, particularly, the most 3′ stem loopstructure, in this region.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the present invention haveat most one mismatch with the target sequence out of 10 nucleotides, andpreferably at most one mismatch out of 20. Alternatively, the antisenseoligomers employed have at least 90% sequence homology, and preferablyat least 95% sequence homology, with the exemplary targeting sequencesas designated herein.

An oligonucleotide analog “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a T_(m) substantially greater than 45°C., preferably at least 50° C., and typically 60° C.-80° C. or higher.Such hybridization preferably corresponds to stringent hybridizationconditions. At a given ionic strength and pH, the T_(m) is thetemperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide. Again, such hybridization may occur with“near” or “substantial” complementary of the antisense oligomer to thetarget sequence, as well as with exact complementarity.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form, by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex” refers to a duplex between an oligonucleotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “base-specific intracellular binding event involving a target RNA”refers to the specific binding of an oligonucleotide analog to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

An “effective amount” of an antisense oligomer, targeted against aninfecting ssRNA virus, is an amount effective to reduce the rate ofreplication of the infecting virus, and/or viral load, and/or symptomsassociated with the viral infection.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of the individual orcell. Treatment includes, but is not limited to, administration of e.g.,a pharmaceutical composition, and may be performed eitherprophylactically, or subsequent to the initiation of a pathologic eventor contact with an etiologic agent. The related term “improvedtherapeutic outcome” relative to a patient diagnosed as infected with aparticular virus, refers to a slowing or diminution in the growth ofvirus, or viral load, or detectable symptoms associated with infectionby that particular virus.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, the oligonucleotide analog preferably has a substantiallyuncharged backbone, as defined below. Alternatively, the antisensecompound may be formulated in a complexed form, such as an agent havingan anionic backbone complexed with cationic lipids or liposomes, whichcan be taken into cells by an endocytotic mechanism. The analog also maybe conjugated, e.g., at its 5′ or 3′ end, to an arginine-rich peptide,e.g., a portion of the HIV TAT protein, or polyarginine, to facilitatetransport into the target host cell as described (Moulton, Nelson et al.2004). Exemplary arginine-rich peptides useful in practicing the presentinvention as listed as SEQ ID NOS: 121-126 in the Sequence Listingtable.

A ssRNA sequence is “capable of forming internal stem-loop secondarystructure” if it can spontaneously, under physiological conditions, formone or more regions of double-stranded (duplex) RNA separated by one ormore regions of single-stranded RNA. The stem and loop in this structurerefers to the duplex RNA region (stem) terminating in a loopedsingle-stranded region. “Internal” refers to the fact that the secondarystructure is contained within the adjacent 5′-terminal regions ofsequence. The stem-loop structure of the 5′-terminal regions of severalof the viruses encompassed by the invention are shown in FIGS. 4A-4E(FIG. 4A—JEV, SEQ ID NO.:2; FIG. 4A—MVEV, SEQ ID NO.:3; FIG. 4A—WNV, SEQID NO.:4; FIG. 4A—YFV, SEQ ID NO.:5; FIG. 4A—DEN2, SEQ ID NO.:6; FIG.4A—HCV, SEQ ID NO.:7; FIG. 4B—PV, SEQ ID NO.:11; FIG. 4B—HEV-A, SEQ IDNO.:12; FIG. 4B—HEV-B, SEQ ID NO.:13; FIG. 4B—HEV-C, SEQ ID NO.:14; FIG.4B—HEV-E, SEQ ID NO.:16; FIG. 4B—BEV, SEQ ID NO.:17; FIG. 4B—HRV-89, SEQID NO.:18; FIG. 4B—HRV-B, SEQ ID NO.:19; FIG. 4B—FMDV, SEQ ID NO.:20;FIG. 4B—HAV, SEQ ID NO.:21; FIG. 4C—TBEV, SEQ ID NO.:8; FIG. 4C—OHFV,SEQ ID NO.:9; FIG. 4C—Powassan, SEQ ID NO.:10; FIG. 4C—FCV, SEQ IDNO.:22; FIG. 4C—CaCV, SEQ ID NO.:23; FIG. 4C—PoCV, SEQ ID NO.:24; FIG.4C—NV, SEQ ID NO.:26; FIG. 4C—RUBV, SEQ ID NO.:28; FIG. 4D—CVNB, SEQ IDNO.:25; FIG. 4D—HEV, SEQ ID NO.:27; FIG. 4D—SARS, SEQ ID NO.:29; FIG.4D—PEDV, SEQ ID NO.:30; FIG. 4D—TGEV, SEQ ID NO.:31; FIG. 4D—BCoV, SEQID NO.:32; FIG. 4D—HCoV-229E, SEQ ID NO.:33; FIG. 4D—MHV, SEQ ID NO.:34;FIG. 4E—PRRSV, SEQ ID NO.:35; FIG. 4E—HAstV, SEQ ID NO.:37; FIG.4E—EEEV, SEQ ID NO.:38; FIG. 4E—WEEV, SEQ ID NO.:39; FIG. 4E—VEEV, SEQID NO.:40). As seen for the West Nile virus (WNV) or HCV, for example(FIG. 4A), the stem-loop structure may comprise a single stem, a singleloop, and non-duplexed end regions. In other cases, e.g., Yellow fevervirus (YFV) or Dengue-2 virus (FIG. 4A), the stem-loop secondarystructure can include two or more double-stranded “stem” regionsinterspersed by non-duplexed regions and including a single loop.

By “disruption of such stem-loop structure” is meant disruption of anyportion of the stem-loop structure in the 5′ terminal region of the RNAviral positive-strand genome, by interfering with duplex RNA formationwithin this region by forming a stable heteroduplex complex between theoligonucleotide analog compound of the invention and duplex-formingsequences within the 5′ terminal region of the virus genome. Rules forthe selection of targeting sequences capable of disrupting secondarystem-loop structure in the 5′-terminal region of a viral genome arediscussed below.

II. Targeted Viruses

The present invention is based on the discovery that effective ofsingle-stranded, positive-sense RNA viruses can be achieved by exposingcells infected with the virus to antisense oligonucleotide analogcompounds (i) targeted against the 5′ end terminal sequences of thepositive-strand viral RNA strand, and in particular, against targetsequences that contribute to stem-loop secondary structure in thisregion, (ii) having physical and pharmacokinetic features which alloweffective interaction between the antisense compound and the viruswithin host cells. In one aspect, the oligomers can be used in treatinga mammalian subject infected with the virus.

The invention targets RNA viruses having genomes that are: (i) singlestranded, (ii) positive polarity, and (iii) less than 32 kb. Thetargeted viruses also synthesize a genomic RNA strand with negativepolarity, the minus-strand or negative-sense RNA, as the first step inviral RNA replication. In particular, targeted viral families includeFlaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae,Coronaviridae, Astroviridae and Hepeviridae families. Targeted virusesorganized by family, genus and species are listed in Table 1. Variousphysical, morphological, and biological characteristics of each of theseeight families, and members therein, can be found, for example, inTextbook of Human Virology, R. Belshe, ed., 2^(nd) Edition, Mosby, 1991and at the Universal Virus Database of the International Committee onTaxonomy of Viruses (www.ncbi.nlm.nih.gov/ICTVdb/index.htm). Some of thekey biological characteristics of each family are summarized below.

A. Flaviviridae. Members of this family include several serious humanpathogens, among them mosquito-borne members of the genus Flavivirusincluding yellow fever virus (YFV), West Nile virus (WNV), Japaneseencephalitis virus (JEV), St. Louis encephalitis virus (SLEV), MurrayValley encephalitis, Kunjin virus, and the four serotypes of denguevirus (DEN1-4).

Tick-borne members of the Flavivirus genus include tick-borneencephalitis virus (TBEV) and related viruses including Omsk hemorrhagicfever virus (OHFV), Louping ill virus, Powassan virus, Kyasanur Forestdisease virus and Alkhurma virus.

The Flaviviridae also includes Hepatitis C virus, a member of the genusHepacivirus.

B. Picornaviridae. This medically important family, whose members infectboth humans and animals, can cause severe paralysis (paralyticpoliomyelitis), aspectic meningitis, hepatitis, pleurodynia,myocarditis, skin rashes, and colds; unapparent infection is common.Several medically important genera are members of this family;Enterovirus including poliovirus (PV) and human enteroviruses (e.g.coxsackie viruses); Hepatovirus which includes hepatitis A virus (HAV);Rhinoviruses; and Aphthoviruses which include the foot-and mouth diseasevirus (FMDV).

Rhinoviruses such as human rhinovirus 89 (HRV-89) and human rhinovirus B(HRV-B) are recognized as the principle cause of the common cold inhumans. Serotypes are designated from 1A to 100. Transmission isprimarily by the aerosol route and the virus replicates in the nose.

Like all positive-sense RNA viruses, the genomic RNA of Picornavirusesis infectious; that is, the genomic RNA is able to direct the synthesisof viral proteins directly, without host transcription events.

C. Caliciviridae. Members of the Caliciviridae infect both humans andanimals. The genus Vesivirus produces disease manifestations in mammalsthat include epithelial blistering and are suspected of being the causeof animal abortion storms and some forms of human hepatitis (non Athrough E) (Smith et al., 1998). Other genera of the Caliciviridaeinclude the Norwalk-like and Sapporo-like viruses, which togethercomprise the human caliciviruses, and the Lagoviruses, which includerabbit hemorrhagic disease virus, a particularly rapid and deadly virus.

The human caliciviruses are the most common cause of viral diarrheaoutbreaks worldwide in adults, as well as being significant pathogens ofinfants (O'Ryan 1992). There are at least five types of humancaliciviruses that inhabit the gastrointestinal tract. The Norwalk virusis a widespread human agent causing acute epidemic gastroenteritis andcauses approximately 10% of all outbreaks of gastroenteritis in man(Murray and al. 1998).

Vesiviruses are now emerging from being regarded as somewhat obscure andhost specific to being recognized as one of the more versatile groups ofviral pathogens known. For example, a single serotype has been shown toinfect a diverse group of 16 different species of animals that include asaltwater fish (opal eye), sea lion, swine, and man.

D. Togaviridae. Members of this family include the mosquito-borneviruses which infect both humans and animals. The family includes thegenera Alphavirus and Rubivirus (rubella). Representative Alphavirusesinclude Sindbus, Western equine encephalomyelitis virus (WEEV), Easternequine encephalitis virus (EEEV) and Venezuelan equine encephalitisvirus (VEEV).

E. Hepatitis E-like Viruses. Hepatitis E virus (HEV) was initiallydescribed in 1987 and first reported in the U.S. in 1991. The virus wasinitially described as a member of the Caliciviridae based on the small,single-stranded RNA character. Some still classify HEV as belonging tothe Caliciviridae, but it has also been recently classified as the onlymember of the Hepeviridae family. Infection appears to be much likehepatitis A viral infection. The disease is an acute viral hepatitiswhich is apparent about 20 days after initial infection, and the virusmay be observed for about 20 days in the serum. Transmission occursthrough contaminated water and geographically the virus is restricted toless developed countries.

F. Coronaviridae, Arteriviridae and Astroviridae. Members of theCoronaviridae include the human coronaviruses that cause 10 to 30% ofcommon colds and other respiratory infections, and murine hepatitisvirus. More recently, the viral cause of severe acute respiratorysyndrome (SARS) has been identified as a coronavirus. The Arteriviridaeinclude two important animal viruses, Equine arteritis virus (EAV) andporcine reproductive and respiratory syndrome virus (PRRSV). TheAstroviridae includes the human astrovirus (HAstV).

Family Genus Virus Flaviviridae Flavivirus St. Louis encephalitis (SLEV)Japanese encephalitis (JEV) Murray Valley encephalitis (MVEV) West Nile(WNV) Yellow fever (YFV) Dengue Types 1–4 (DEN1–4) Tick-borneencephalitis (TBEV) Omsk hemorrhagic fever (OHFV) Powassan HepacivirusHepatitis C (HCV) Picornaviridae Enterovirus Poliovirus (PV) Humanenterovirus A (HEV-A) Human enterovirus B (HEV-B) Human enterovirus C(HEV-C) Human enterovirus D (HEV-D) Human enterovirus E (HEV-E) Bovineenterovirus (BEV) Rhinovirus Human Rhinovirus B (HRV-B) Human Rhinovirus89 (HRV-89) Apthovirus Foot and mouth disease (FMDV) HepatovirusHepatitis A (HAV) Caliciviridae Vesivirus Feline calicivirus (FCV)Canine calicivirus (CaCV) Porcine enteric calcivirus (PoCV) Calicivirusstrain NB (CVNB) Norovirus Norwalk(NV) Hepeviridae Hepevirus Hepatitis E(HEV) Togaviridae Rubivirus Rubella (RUBV) Alphavirus Eastern equineencephalitis (EEEV) Western equine encephalitis (WEEV) Venezuelan equineencephalitis (VEEV) Coronaviridae Coronavirus Porcine epidemic diarrhea(PEDV) Transmissible gastroenteritis (TGEV) SARS coronavirus (SARS-CoV)Bovine coronavirus (BCoV) Human coronavirus 229E (HCoV- 229E) Murinehepatitis (MHV) Arteriviridae Arterivirus Equine arteritis (EAV) Porcinerespiratory and reproductive syndrome (PRRSV) Astroviridae MamastrovirusHuman astrovirus (HAstV)

III. Viral Target Regions

Single-stranded, positive-sense RNA viruses, like all RNA viruses, areunique in their ability to synthesize RNA on an RNA template. To achievethis task they encode and induce the synthesis of a unique RNA-dependentRNA polymerases (RdRp) and possibly other proteins which bindspecifically to the 3′ and 5′ end terminal untranslated regions (UTRs)of viral RNA. Since viral RNAs are linear molecules, RdRps have toemploy unique strategies to initiate de novo RNA replication whileretaining the integrity of the 5′ end of their genomes. It is generallyaccepted that positive-strand (+strand) viral RNA replication proceedsvia the following pathway:+strand RNA→−strand RNA synthesis→−RF→+strand RNA synthesis

where “−strand RNA” is negative-sense or minus-strand RNA complementaryto the “+strand RNA” and “RF” (replicative form) is double-stranded RNA.The minus-strand RNA is used as a template for replication of multiplecopies of positive-strand RNA which is destined for either translationinto viral proteins or incorporation into newly formed virions. Theratio of positive to minus-strand RNA in poliovirus-infected cells isapproximately 50:1 in Hepatitis C-infected cells indicating eachminus-strand RNA serves as a template for the synthesis of manypositive-strand RNA molecules.

The present invention is based on the discovery that several classes ofpositive-strand RNA viruses can be effectively inhibited by exposing theviruses to an anti-sense compound capable of binding to a sequencewithin the ′5 UTR of the virus positive strand, and in particular, to asequence designed to disrupt one or more of the cis-acting elements(stem-loop structures) within the 5′ UTR.

Therefore, as a first step in identifying an effective target region(the sequence in the positive strand 5′-UTR to which the antisensecompound will bind), one identifies those region within the 5′-UTR whichare involved in stem-loop secondary structure. This may be done, forexample, by computer-assisted secondary structure predictions which arebased on a search for the minimal free energy state of the input RNAsequence (Zuker 2003). When this analysis is applied to the terminal 40bases of the 5′-UTR region of various target viruses, the secondarystructures or stem loops shown in FIG. 4A-4E (FIG. 4A—JEV, SEQ ID NO.:2;FIG. 4A—MVEV, SEQ ID NO.:3; FIG. 4A—WNV, SEQ ID NO.:4; FIG. 4A—YFV, SEQID NO.:5; FIG. 4A—DEN2, SEQ ID NO.:6; FIG. 4A—HCV, SEQ ID NO.:7; FIG.4B—PV, SEQ ID NO.:11; FIG. 4B—HEV-A, SEQ ID NO.:12; FIG. 4B—HEV-B, SEQID NO.:13; FIG. 4B—HEV-C, SEQ ID NO.:14; FIG. 4B—HEV-E, SEQ ID NO.:16;FIG. 4B—BEV, SEQ ID NO.:17; FIG. 4B—HRV-89, SEQ ID NO.:18; FIG.4B—HRV-B, SEQ ID NO.:19; FIG. 4B—FMDV, SEQ ID NO.:20; FIG. 4B—HAV, SEQID NO.:21; FIG. 4C—TBEV, SEQ ID NO.:8; FIG. 4C—OHFV, SEQ ID NO.:9; FIG.4C—Powassan, SEQ ID NO.:10; FIG. 4C—FCV, SEQ ID NO.:22; FIG. 4C—CaCV,SEQ ID NO.:23; FIG. 4C—PoCV, SEQ ID NO.:24; FIG. 4C—NV, SEQ ID NO.:26;FIG. 4C—RUBV, SEQ ID NO.:28; FIG. 4D—CVNB, SEQ ID NO.:25; FIG. 4D—HEV,SEQ ID NO.:27; FIG. 4D—SARS, SEQ ID NO.:29; FIG. 4D—PEDV, SEQ ID NO.:30;FIG. 4D—TGEV, SEQ ID NO.:31; FIG. 4D—BCoV, SEQ ID NO.:32; FIG.4D—HCoV-229E, SEQ ID NO.:33; FIG. 4D—MHV, SEQ ID NO.:34; FIG. 4E—PRRSV,SEQ ID NO.:35; FIG. 4E—HAstV, SEQ ID NO.:37; FIG. 4E—EEEV, SEQ IDNO.:38; FIG. 4E—WEEV, SEQ ID NO.:39; FIG. 4E—VEEV, SEQ ID NO.:40) areobtained. As seen, regions of secondary structure (forming thecis-acting elements) are found typically in the terminal 20-25 bases,but in many cases, in bases up to position 40. Therefore, the preferredtarget sequences are the 5′ end terminal regions of the positive-strandRNA that include the end-most 40 nucleotides, typically the 5′ terminal5-35 nucleotides. Preferred target regions include those bases involvedin secondary structure in these regions, as indicated in FIGS. 4A-4D(FIG. 4A—JEV, SEQ ID NO.:2; FIG. 4A—MVEV, SEQ ID NO.:3; FIG. 4A—WNV, SEQID NO.:4; FIG. 4A—YFV, SEQ ID NO.:5; FIG. 4A—DEN2, SEQ ID NO.:6; FIG.4A—HCV, SEQ ID NO.:7; FIG. 4B—PV, SEQ ID NO.:11; FIG. 4B—HEV-A, SEQ IDNO.:12; FIG. 4B—HEV-B, SEQ ID NO.:13; FIG. 4B—HEV-C, SEQ ID NO.:14; FIG.4B—HEV-E, SEQ ID NO.:16; FIG. 4B—BEV, SEQ ID NO.:17; FIG. 4B—HRV-89, SEQID NO.:18; FIG. 4B—HRV-B, SEQ ID NO.:19; FIG. 4B—FMDV, SEQ ID NO.:20;FIG. 4B—HAV, SEQ ID NO.:21; FIG. 4C—TBEV, SEQ ID NO.:8; FIG. 4C—OHFV,SEQ ID NO.:9; FIG. 4C—Powassan, SEQ ID NO.:10; FIG. 4C—FCV, SEQ IDNO.:22; FIG. 4C—CaCV, SEQ ID NO.:23; FIG. 4C—PoCV, SEQ ID NO.:24; FIG.4C—NV, SEQ ID NO.:26; FIG. 4C—RUBV, SEQ ID NO.:28; FIG. 4D—CVNB, SEQ IDNO.:25; FIG. 4D—HEV, SEQ ID NO.:27; FIG. 4D—SARS, SEQ ID NO.:29; FIG.4D—PEDV, SEQ ID NO.:30; FIG. 4D—TGEV, SEQ ID NO.:31; FIG. 4D—BCoV, SEQID NO.:32; FIG. 4D—HCoV-229E, SEQ ID NO.:33; FIG. 4D—MHV). Inparticular, the targeting sequence contains a sequence of at least 12bases that are complementary to the 5′-end region of the positive-strandRNA, and are selected such that hybridization of the compound to the RNAis effective to disrupt stem-loop secondary structure in this region,preferably the 5′-end most stem-loop secondary structure. By way ofexample, FIG. 4A shows secondary structure of viral-genome sequencesthat are available from well known sources, such as the NCBI Genbankdatabases. Alternatively, a person skilled in the art can find sequencesfor many of the subject viruses in the open literature, e.g., bysearching for references that disclose sequence information ondesignated viruses. Once a complete or partial viral sequence isobtained.

The general genomic organization of each of the eight virus families isdiscussed below, followed by exemplary target sequences obtained forselected members (genera, species or strains) within each family.

A. Picornaviridae. Typical of the picornaviruses, the human rhinovirus89 genome (FIG. 3A) is a single molecule of single-stranded,positive-sense, polyadenylated RNA of approximately 7.2 kb. The genomeincludes a long 618 nucleotide UTR which is located upstream of thefirst polyprotein, a single ORF, and a VPg (viral genome linked) proteincovalently attached to its 5′ end. The ORF is subdivided into twosegments, each of which encodes a polyprotein. The first segment encodesa polyprotein that is cleaved subsequently to form viral proteins VP1 toVP4, and the second segment encodes a polyprotein which is the precursorof viral proteins including a protease and a polymerase. The ORFterminates in a polyA termination sequence.

B. Caliciviridae. FIG. 3B shows the genome of a calicivirus; in thiscase the Norwalk virus. The genome is a single molecule of infectious,single stranded, positive-sense RNA of approximately 7.6 kb. As shown,the genome includes a small UTR upstream of the first open reading framewhich is unmodified. The 3′ end of the genome is polyadenylated. Thegenome includes three open reading frames. The first open reading frameencodes a polyprotein, which is subsequently cleaved to form the viralnon-structural proteins including a helicase, a protease, an RNAdependent RNA polymerase, and “VPg”, a protein that becomes bound to the5′ end of the viral genomic RNA (Clarke and Lambden, 2000). The secondopen reading frame codes for the single capsid protein, and the thirdopen reading frame codes for what is reported to be a structural proteinthat is basic in nature and probably able to associate with RNA.

C. Togaviridae. FIG. 3C shows the structure of the genome of atogavirus, in this case, a rubella virus of the Togavirus genus. Thegenome is a single linear molecule of single-stranded, positive-senseRNA of approximately 9.8 kb, which is infectious. The 5′ end is cappedwith a 7-methylG molecule and the 3′ end is polyadenylated. Full-lengthand subgenomic messenger RNAs have been demonstrated, and posttranslational cleavage of polyproteins occurs during RNA replication.The genome also includes two open reading frames. The first open readingframe encodes a polyprotein which is subsequently cleaved into fourfunctional proteins, nsP1 to nsP4. The second open reading frame encodesthe viral capsid protein and three other viral proteins, PE2, 6K and E1.

D. Flaviviridae. FIG. 3D shows the structure of the genome of thehepatitis C virus of the Hepacivirus genus. The HCV genome is a singlelinear molecule of single-stranded, positive-sense RNA of about 9.6 kband contains a 341 nucleotide 5′ UTR. The 5′ end is capped with anm⁷GppAmp molecule, and the 3′ end is not polyadenylated. The genomeincludes only one open reading frame which encodes a precursorpolyprotein separable into six structural and functional proteins.

E. Coronaviridae. FIG. 3E shows the genome structure of humancoronavirus 229E. This coronovirus has a large genome of approximately27.4 kb that is typical for the Coronoviridae and a 292 nucleotide 5′UTR. The 5′-most ORF of the viral genome is translated into a largepolyprotein that is cleaved by viral-encoded proteases to releaseseveral nonstructural proteins, including an RdRp and a helicase. Theseproteins, in turn, are responsible for replicating the viral genome aswell as generating nested transcripts that are used in the synthesis ofother viral proteins.

GenBank references for exemplary viral nucleic acid sequencesrepresenting the 5′ end terminal, positive-strand sequences for thefirst (most 5′-end) 40 bases for corresponding viral genomes are listedin Table 2 below. The nucleotide sequence numbers in Table 2 are derivedfrom the Genbank reference for the positive-strand RNA. It will beappreciated that these sequences are only illustrative of othersequences in the five virus families, as may be available from availablegene-sequence databases of literature or patent resources. The sequencesbelow, identified as SEQ ID NOS: 1-40, are also listed in SequenceListing, Table 4, at the end of the specification.

The target sequences in Table 2 are the first 40 bases at the 5′terminal ends of the positive-strands sequences of the indicated viralRNAs. The sequences shown are in the 5′ to 3′ orientation so the 3′terminal nucleotide is at the end of the listed sequence. The basedesignation “N” indicates the nucleotides at these positions arepresently unknown. The region within each sequence that is associatedwith stem-loop secondary structure can be seen from the predictedsecondary structures in these sequences, shown in FIGS. 4A-4D (FIG.4A—JEV, SEQ ID NO.:2; FIG. 4A—MVEV, SEQ ID NO.:3; FIG. 4A—WNV, SEQ IDNO.:4; FIG. 4A—YFV, SEQ ID NO.:5; FIG. 4A—DEN2, SEQ ID NO.:6; FIG.4A—HCV, SEQ ID NO.:7; FIG. 4B—PV, SEQ ID NO.:11; FIG. 4B—HEV-A, SEQ IDNO.:12; FIG. 4B—HEV-B, SEQ ID NO.:13; FIG. 4B—HEV-C, SEQ ID NO.:14; FIG.4B—HEV-E, SEQ ID NO.:16; FIG. 4B—BEV, SEQ ID NO.:17; FIG. 4B—HRV-89, SEQID NO.:18; FIG. 4B—HRV-B, SEQ ID NO.:19; FIG. 4B—FMDV, SEQ ID NO.:20;FIG. 4B—HAV, SEQ ID NO.:21; FIG. 4C—TBEV, SEQ ID NO.:8; FIG. 4C—OHFV,SEQ ID NO.:9; FIG. 4C—Powassan, SEQ ID NO.:10; FIG. 4C—FCV, SEQ IDNO.:22; FIG. 4C—CaCV, SEQ ID NO.:23; FIG. 4C—PoCV, SEQ ID NO.:24; FIG.4C—NV, SEQ ID NO.:26; FIG. 4C—RUBV, SEQ ID NO.:28; FIG. 4D—CVNB, SEQ IDNO.:25; FIG. 4D—HEV, SEQ ID NO.:27; FIG. 4D—SARS, SEQ ID NO.:29; FIG.4D—PEDV, SEQ ID NO.:30; FIG. 4D—TGEV, SEQ ID NO.:31; FIG. 4D—BCoV, SEQID NO.:32; FIG. 4D—HCoV-229E, SEQ ID NO.:33; FIG. 4D—MHV).

TABLE 2 Exemplary 5′ End Terminal Viral Nucleic Acid Target SequencesSEQ Gen-Bank ID Virus No. Target Sequence (5′ to 3′) NO. St. LouisM18929 GNNGATGTTCGCGTCGGTGAGCGGAGAGGAA encephalitis ACAGATTTC (SLEV)Japanese NC 001437 AGAAGTTTATCTGTGTGAACTTCTTGGCTTAG 2 encephalitis (JEV)TATCGTTG Murray Valley NC 000943 AGACGTTCATCTGCGTGAGCTTCCGATCTCA 3encephalitis GTATTGTTT (MVEV) West Nile NC 001563AGTAGTTCGCCTGTGTGAGCTGACAAACTTA 4 (WNV) GTAGTGTTT Yellow Fever NC 002031AGTAAATCCTGTGTGCTAATTGAGGTGCATT 5 (YFV) GGTCTGCAA Dengue - Type 2 M20558AGTTGTTAGTCTACGTGGACCGACAAAGACA 6 (DEN2) GATTCTTTG Hepatitis C NC 004102GCCAGCCCCCTGATGGGGGCGACACTCCACC 7 (HCV) ATGAATCAC Tick-borne enceph-NC 001672 AGATTTTCTTGCACGTGCATGCGTTTGCTTCG 8 alitis virus (TBEV)GACAGCAT Omsk hemorrhagic NC 005062 AGATTTTCTTGCACGTGCGTGCGCTTGCTTCA 9fever (OHFV) GACAGCAA Powassan NC 003687AGATTTTCTTGCACGTGTGTGCGGGTGCTTTA 10 GTCAGTGT Poliovirus- NC 002058TTAAAACAGCTCTGGGGTTGTACCCACCCCA 11 Mahoney strain GAGGCCCAC (PV)Human enterovirus NC 001612 TTAAAACAGCCTGTGGGTTGTACCCACCCAC 12 AAGGGCCCAC (HEV-A) Human enterovirus NC 001472TTAAAACAGCCTGTGGGTTGTTCCCACCCAC 13 B AGGCCCATT (HEV-B) Human enterovirusNC 001428 TTAAAACAGCTCTGGGGTTGCTCCCACCCCA 14 C GAGGCCCAC (HEV-C)Human enterovirus NC 001430 TTAAAACAGCTCTGGGGTTGTTCCCACCCCA 15 DGAGGCCCAC (HEV-D) Human enterovirus NC 003988GAGTGTTCCCACCCAACAGGCCCACTGGGTG 16 E TTGTACTCT (HEV-E)Bovine enterovirus NC 001859 TTAAAACAGCCTGGGGGTTGTACCCACCCCT 17 (BEV)GGGGCCCAC Human rhinovirus NC 001617 TTAAAACTGGGAGTGGGTTGTTCCCACTCAC 1889 TCCACCCAT (HRV-89) Human rhinovirus NC 001490TTAAAACAGCGGATGGGTATCCCACCATTCG 19 B ACCCATTGG (HRV-B) Foot-and-mouthAY593768 TTGAAAGGGGGCGCTAGGGTTTCACCCCTAG 20 disease virus CATGCCAAC(FMDV) Hepatitis A NC 001489 TTCAAGAGGGGTCTCCGGGAATTTCCGGAGT 21 (HAV)CCCTCTTGG Feline calicivirus NC 001481 GTAAAAGAAATTTGAGACAATGTCTCAAACT22 (FCV) CTGAGCTTC Canine calicivirus NC 004542GTTAATGAGAAATGGCTTCTGCCATCGCTCT 23 (CaCV) CTCGAGCTC Porcine entericNC 000940 GTGATCGTGATGGCTAATTGCCGTCCGTTGC 24 calicivirus CTATTGGGC(PoCV) Calicivirus strain NC 004064 GTGATTTAATTATAGAGAGATAGTGACTTTC 25NB CVNB ACTTTTCTT Norwalk (NV) NC 001959 GTGAATGATGATGGCGTCAAAAGACGTCGTT26 CCTACTGCT Hepatitis E NC 001434 GCCATGGAGGCCCATCAGTTTATTAAGGCTC 27(HEV) CTGGCATCA Rubella (RUBV) NC 001545 ATGGAAGCTATCGGACCTCGCTTAGGACTCC28 CATTCCCAT SARS coronavirus NC 004718 ATATTAGGTTTTTACCTACCCAGGAAAAGCC29 (SARS-CoV) AACCAACCT Porcine epidemic NC 003436ACTTAAAAAGATTTTCTATCTACGGATAGTTA 30 diarrhea (PEDV) GCTCTTTTTransmissible NC 002306 ACTTTTAAAGTAAAGTGAGTGTAGCGTGGCT 31gastroenteritis ATATCTCTT (TGEV) Bovine coronavirus NC 003045GATTGCGAGCGATTTGCGTGCGTGCATCCCG 32 (BCoV) CTTCACTGA Human corona-NC 002645 ACTTAAGTACCTTATCTATCTACAGATAGAAA 33 virus 229E AGTTGCTT(HCoV-229E) Murine Hepatitis NC 001846 TATAAGAGTGATTGGCGTCCGTACGTACCCT34 (MHV) CTCAACTCT Porcine repro AF 176348ATGACGTATAGGTGTTGGCTCTATGCCTTGG 35 ductive and CATTTGTATrespiratory syn- drome (PRRSV) Equine arteritis NC 002532GCTCGAAGTGTGTATGGTGCCATATACGGCT 36 (EAV) CACCACCAT Human astro-virusNC 001943 CCAAGAGGGGGGTGGTGATTGGCCTTTGGCT 37 (HAstV) TATCAGTGTEastern equine NC 003899 ATAGGGTACGGTGTAGAGGCAACCACCCTAT 38 encephalitisTTCCACCTA (EEEV) Western equine NC 003908ACCCTACAAACTAATCGATCCAATATGGAAA 39 encephalomyelits GAATTCACG (WEEV)Venezuelan equine NC 001449 ATGGGCGGCGCAAGAGAGAAGCCCAAACCAA 40enceph-alitis TTACCTACC (VEEV)

To select a targeting sequence, one looks for a sequence that, whenhybridized to a complementary sequence in the 5′-end region of thepositive-strand RNA (SEQ ID NOS: 1-40), will be effective to disruptstem-loop secondary structure in this region, and preferably, theinitial stem structure in the region. By way of example, a suitabletargeting sequence for the West Nile Virus (WNV in FIG. 4A) is asequence that will disrupt the stem loop structure shown in the figure.Three general classes of sequences would be suitable (exemplary 12-14base targeting sequences are shown for illustrative purposes):

-   (1) a sequence such as 5′-ACAGGCGAACTACT-3′ (SEQ ID NO:134) that    targets the most 5′ bases (1-14) of the stem and surrounding bases;-   (2) a sequence such as 5′-GTCAGCTCACAC-3′SEQ ID NO:135) that targets    the complementary bases of the stem and surrounding bases (13-24);-   (3) a sequence such as 6-GCTCACACAGGCGA-3′ (SEQ ID NO:136) that    targets a portion of one or both “sides” of a stem loop and    surrounding bases (7-20); typically, the sequence should disrupt all    but at least 2-4 of the paired bases forming the stem structure.

It will be appreciated how this selection procedure can be applied tothe other sequences shown in Table 2, For example, for the yellow fevervirus (YFV) shown in FIG. 4A, exemplary 14-18 base sequences patternedafter the three general classes above, might include:

-   (1) a sequence such as 5′-GCACACAGGATTTACT-3′ (SEQ ID NO:137) that    targets the most 5′ bases (1-16) of the intial stem and surrounding    bases;-   (2) a sequence such as 5′-GTCCAATGCACCTC-3′ (SEQ ID NO:138) that    targets the complementary bases of the initial stem and surrounding    bases (22-35);-   (3) a sequence such as 5′-CAATGCACCTCAATTAGC-3′ (SEQ ID NO:139) that    targets a portion of both sides of a stem and surrounding bases    (15-32).

It will be understood that targeting sequences so selected can be madeshorter, e.g., 12 bases, or longer, e.g., 20 bases, and include a smallnumber of mismatches, as long as the sequence is sufficientlycomplementary to disrupt the stem structure(s) upon hybridization withthe target, and forms with the virus positive-strand, a heteroduplexhaving a Tm of 45° C. or greater.

More generally, the degree of complementarity between the target andtargeting sequence is sufficient to form a stable duplex. The region ofcomplementarity of the antisense oligomers with the target RNA sequencemay be as short as 8-11 bases, but is preferably 12-15 bases or more,e.g. 12-20 bases, or 12-25 bases. An antisense oligomer of about 14-15bases is generally long enough to have a unique complementary sequencein the viral genome. In addition, a minimum length of complementarybases may be required to achieve the requisite binding T_(m), asdiscussed below.

Oligomers as long as 40 bases may be suitable, where at least theminimum number of bases, e.g., 8-11, preferably 12-15 bases, arecomplementary to the target sequence. In general, however, facilitatedor active uptake in cells is optimized at oligomer lengths less thanabout 30, preferably less than 25, and more preferably 20 or fewerbases. For PMO oligomers, described further below, an optimum balance ofbinding stability and uptake generally occurs at lengths of 14-22 bases.

The oligomer may be 100% complementary to the viral nucleic acid targetsequence, or it may include mismatches, e.g., to accommodate variants,as long as a heteroduplex formed between the oligomer and viral nucleicacid target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Oligomer backbones which are less susceptible to cleavage bynucleases are discussed below. Mismatches, if present, are lessdestabilizing toward the end regions of the hybrid duplex than in themiddle. The number of mismatches allowed will depend on the length ofthe oligomer, the percentage of G:C base pairs in the duplex, and theposition of the mismatch(es) in the duplex, according to well understoodprinciples of duplex stability. Although such an antisense oligomer isnot necessarily 100% complementary to the viral nucleic acid targetsequence, it is effective to stably and specifically bind to the targetsequence, such that a biological activity of the nucleic acid target,e.g., expression of viral protein(s), is modulated.

The stability of the duplex formed between the oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107. Each antisenseoligomer should have a binding T_(m), with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. T_(m)'s in the range 60-80° C. or greaterare preferred. According to well known principles, the T_(m) of anoligomer compound, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high T_(m) (50° C. or greater) at a length of 20bases or less are generally preferred over those requiring greater than20 bases for high T_(m) values.

Table 3 below shows exemplary targeting sequences, in a 5′-to-3′orientation, that are complementary to upstream and downstream portionsof the 5′ terminal 40 base regions of the positive strand of the virusesindicated. The sequences here provide a collection of targeting sequenceor sequences from which targeting sequences may be selected, accordingto the general class rules discussed above. Thus, for example, inselecting a target against St. Louis encephalitis virus, one mightselect either SEQ ID NOS: 40 or 41, or a portion of either sequenceeffective to block secondary structure formation in the virus' 5′terminal UTR.

TABLE 3 Exemplary Antisense Sequences Targeting the 5′End Terminal Positive-Strand Regions SEQ GenBank ID Virus Acc. No. Ncts.Sequences (5′ to 3′) NO. St. Louis encephalitis M16614 1-18ACCGACGCGAACATCNNC 41 11-30 TCCTCTCCGCTCACCGACGC 42Japanese encephalitis NC 001437 1-18 TCACACAGATAAACTTCT 43 11-30AAGCCAAGAAGTTCACACAG 44 Murray Valley NC 000943 1-18 TCACGCAGATGAACGTCT45 encephalitis 11-30 GAGATCGGAAGCTCACGCAG 46 West Nile NC 001563 1-20GCTCACACAGGCGAACTACT 47 11-31 TAAGTTTGTCAGCTCACACAG 48 Yellow FeverNC 002031 1-22 CAATTAGCACACAGGATTTACT 49 21-40 TTGCAGACCAATGCACCTCA 50Dengue -Type 2 M20558 1-20 GTCCACGTAGACTAACAACT 51 11-30GTCTTTGTCGGTCCACGTAG 52 Hepatitis C NC 004102 1-17 CCCATCAGGGGGCTGGC 5310-29 TGGAGTGTCGCCCCCATCAG 54 Tick-borne NC 001672 1-20ATGCACGTGCAAGAAAATCT 55 encephalitis 21-40 ATGCTGTCCGAAGCAAACGC 56Omsk hemorrhagic NC 005062 1-21 CACGCACGTGCAAGAAAATCT 57 fever 13-32TGAAGCAAGCGCACGCACGT 58 Powassan NC 003687 1-20 ACACACGTGCAAGAAAATCT 5921-40 ACACTGACTAAAGCACCCGC 60 Poliovirus-Mahoney NC 002058 1-24GGTACAACCCCAGAGCTGTTTTAA 61 strain 21-40 GTGGGCCTCTGGGGTGGGTA 62Human enterovirus A NC 001612 1-20 CAACCCACAGGCTGTTTTAA 63 21-40GTGGGCCCTGTGGGTGGGTA 64 Human enterovirus B NC 001472 1-20CAACCCACAGGCTGTTTTAA 65 21-40 AATGGGCCTGTGGGTGGGAA 66Human enterovirus C NC 001428 1-20 CAACCCCAGAGCTGTTTTAA 67 21-40GTGGGCCTCTGGGGTGGGAG 68 Human enterovirus D NC 001430 1-20CAACCCCAGAGCTGTTTTAA 69 21-40 GTGGGCCTCTGGGGTGGGAA 70Human enterovirus E NC 003988 1-20 CCTGTTGGGTGGGAACACTC 71 21-40AGAGTACAACACCCAGTGGG 72 Bovine enterovirus NC 001859 1-20CAACCCCCAGGCTGTTTTAA 73 21-40 GTGGGCCCCAGGGGTGGGTA 74Human rhinovirus 89 NC 001617 1-20 CAACCCACTCCCAGTTTTAA 75 21-40ATGGGTGGAGTGAGTGGGAA 76 Human rhinovirus B NC 001490 1-20ATACCCATCCGCTGTTTTAA 77 21-40 CCAATGGGTCGAATGGTGGG 78 Foot-and-mouthAY593768 1-21 AACCCTAGCGCCCCCTTTCAA 79 disease 21-40GTTGGCATGCTAGGGGTGAA 80 Hepatitis A NC 001489 1-20 TCCCGGAGACCCCTCTTGAA81 21-40 CCAAGAGGGACTCCGGAAAT 82 Feline calicivirus NC 001481 1-20TTGTCTCAAATTTCTTTTAC 83 21-40 GAAGCTCAGAGTTTGAGACA 84 Canine calicivirusNC 004542 1-20 AGAAGCCATTTCTCATTAAC 85 21-40 GAGCTCGAGAGAGCGATGGC 86Porcine enteric NC 000940 1-20 CAATTAGCCATCACGATCAC 87 calicivirus 13-32GGCAACGGACGGCAATTAGC 88 Calicivirus strain NB NC 004064 1-20TCTCTCTATAATTAAATCAC 89 11-30 AAAGTCACTATCTCTCTATA 90 Norwalk NC 0019591-20 TTGACGCCATCATCATTCAC 91 21-40 AGCAGTAGGAACGACGTCTT 92 Hepatitis ENC 001434 1-20 AACTGATGGGCCTCCATGGC 93 21-40 TGATGCCAGGAGCCTTAATA 94Rubella NC 001545 1-20 CGAGGTCCGATAGCTTCCAT 95 21-40ATGGGAATGGGAGTCCTAAG 96 SARS coronavirus NC 004718 1-20GGTAGGTAAAAACCTAATAT 97 TOR2 21-40 AGGTTGGTTGGCTTTTCCTG 98Porcine epidemic NC 003436 1-20 GATAGAAAATCTTTTTAAGT 99 diarrhea 21-40AAAAGAGCTAACTATCCGTA 100 Transmissible NC 002306 1-20ACTCACTTTACTTTAAAAGT 101 gastroenteritis 11-30 GCCACGCTACACTCACTTTA 102Bovine coronavirus NC 003045 1-20 CACGCAAATCGCTOGCAATC 103 21-40TCAGTGAAGCGGGATGCACG 104 Human coronavirus NC 002645 1-20GATAGATAAGGTACTTAAGT 105 229E 21-40 AAGCAACTTTTCTATCTGTA 106Murine Hepatitis NC 001846 1-21 CGGACGCCAATCACTCTTATA 107 18-39GAGTTGAGAGGGTACGTACGGA 108 Porcine reproductive & AF176348 5-25CATAGAGCCAACACCTATACG 109 respiratory syndrome 21-40ATACAAATGCCAAGGCATAG 110 Equine arteritis NC 002532 1-20GCACCATACACACTTCGAGC 111 21-40 ATGGTGGTGAGCCGTATATG 112 Human astrovirusNC 001943 1-20 AATCACCACCCCCCTCTTGG 113 11-30 GCCAAAGGCCAATCACCACC 114Eastern equine NC 003899 1-20 GCCTCTACACCGTACCCTAT 115 encephalitis21-40 TAGGTGGAAATAGGGTGGTT 116 Western equine NC 003908 1-20GATCGATTAGTTTGTAGGGT 117 encephalomyelitis 21-40 CGTGAATTCTTTCCATATTG118 Venezuelan equine NC 001449 1-20 TTCTCTCTTGCGCCGCCCAT 119encephalitis 21-40 GGTAGGTAATTGGTTTGGGC 120

IV. Antisense Oligonucleotide Analog Compounds

A. Properties

As detailed above, the antisense oligonucleotide analog compound (theterm “antisense” indicates that the compound is targeted against thevirus' sense or positive-sense strand RNA) has a base sequence targetinga region of the 5′ end 40 bases that are associated with secondarystructure in the negative-strand RNA. In addition, the oligomer is ableto effectively target infecting viruses, when administered to a hostcell, e.g. in an infected mammalian subject. This requirement is metwhen the oligomer compound (a) has the ability to be actively taken upby mammalian cells, and (b) once taken up, form a duplex with the targetssRNA with a T_(m) greater than about 45° C.

As will be described below, the ability to be taken up by cells requiresthat the oligomer backbone be substantially uncharged, and, preferably,that the oligomer structure is recognized as a substrate for active orfacilitated transport across the cell membrane. The ability of theoligomer to form a stable duplex with the target RNA will also depend onthe oligomer backbone, as well as factors noted above, the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases promotes survival and ultimate delivery of the agentto the cell cytoplasm.

Below are disclosed methods for testing any given, substantiallyuncharged backbone for its ability to meet these requirements.

A1. Active or Facilitated Uptake by Cells

The antisense compound may be taken up by host cells by facilitated oractive transport across the host cell membrane if administered in free(non-complexed) form, or by an endocytotic mechanism if administered incomplexed form.

In the case where the agent is administered in free form, the antisensecompound should be substantially uncharged, meaning that a majority ofits intersubunit linkages are uncharged at physiological pH. Experimentscarried out in support of the invention indicate that a small number ofnet charges, e.g., 1-2 for a 15- to 20-mer oligomer, can in fact enhancecellular uptake of certain oligomers with substantially unchargedbackbones. The charges may be carried on the oligomer itself, e.g., inthe backbone linkages, or may be terminal charged-group appendages.Preferably, the number of charged linkages is no more than one chargedlinkage per four uncharged linkages. More preferably, the number is nomore than one charged linkage per ten, or no more than one per twenty,uncharged linkages. In one embodiment, the oligomer is fully uncharged.

An oligomer may also contain both negatively and positively chargedbackbone linkages, as long as opposing charges are present inapproximately equal number. Preferably, the oligomer does not includeruns of more than 3-5 consecutive subunits of either charge. Forexample, the oligomer may have a given number of anionic linkages, e.g.phosphorothioate or N3′→P5′ phosphoramidate linkages, and a comparablenumber of cationic linkages, such as N,N-diethylenediaminephosphoramidates (Dagle, 2000). The net charge is preferably neutral orat most 1-2 net charges per oligomer.

In addition to being substantially or fully uncharged, the antisenseagent is preferably a substrate for a membrane transporter system (i.e.a membrane protein or proteins) capable of facilitating transport oractively transporting the oligomer across the cell membrane. Thisfeature may be determined by one of a number of tests for oligomerinteraction or cell uptake, as follows.

A first test assesses binding at cell surface receptors, by examiningthe ability of an oligomer compound to displace or be displaced by aselected charged oligomer, e.g., a phosphorothioate oligomer, on a cellsurface. The cells are incubated with a given quantity of test oligomer,which is typically fluorescently labeled, at a final oligomerconcentration of between about 10-300 nM. Shortly thereafter, e.g.,10-30 minutes (before significant internalization of the test oligomercan occur), the displacing compound is added, in incrementallyincreasing concentrations. If the test compound is able to bind to acell surface receptor, the displacing compound will be observed todisplace the test compound. If the displacing compound is shown toproduce 50% displacement at a concentration of 10× the test compoundconcentration or less, the test compound is considered to bind at thesame recognition site for the cell transport system as the displacingcompound.

A second test measures cell transport, by examining the ability of thetest compound to transport a labeled reporter, e.g., a fluorescencereporter, into cells. The cells are incubated in the presence of labeledtest compound, added at a final concentration between about 10-300 nM.After incubation for 30-120 minutes, the cells are examined, e.g., bymicroscopy, for intracellular label. The presence of significantintracellular label is evidence that the test compound is transported byfacilitated or active transport.

The antisense compound may also be administered in complexed form, wherethe complexing agent is typically a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to any net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components, are well known. Forexample, the liposomal composition Lipofectin® (Felgner et al., 1987),containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies.

The antisense compound may also be administered in conjugated form withan arginine-rich peptide linked covalently to the 5′ or 3′ end of theantisense oligomer. The peptide is typically 8-16 amino acids andconsists of a mixture of arginine, and other amino acids includingphenyalanine and cysteine. The use of arginine-rich peptide-PMOconjugates can be used to enhance cellular uptake of the antisenseoligomer (See, e.g. (Moulton, Nelson et al. 2004).

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12): 1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., ANTISENSE OLIGONUCLEOTIDES: A NEWTHERAPEUTIC PRINCIPLE, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Alternatively, and according to another aspect of the invention, therequisite properties of oligomers with any given backbone can beconfirmed by a simple in vivo test, in which a labeled compound isadministered to an animal, and a body fluid sample, taken from theanimal several hours after the oligomer is administered, assayed for thepresence of heteroduplex with target RNA. This method is detailed insubsection D below.

A2. Substantial Resistance to RNaseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides. (See e.g., Agrawal et al.,1990; Bonham et al., 1995; and Boudvillain et al., 1997). In the first,a heteroduplex formed between the oligonucleotide and the viral RNA actsas a substrate for RNaseH, leading to cleavage of the viral RNA.Oligonucleotides belonging, or proposed to belong, to this class includephosphorothioates, phosphotriesters, and phosphodiesters (unmodified“natural” oligonucleotides). Such compounds expose the viral RNA in anoligomer:RNA duplex structure to hydrolysis by RNaseH, and thereforeloss of function.

A second class of oligonucleotide analogs, termed “steric blockers” or,alternatively, “RNaseH inactive” or “RNaseH resistant”, have not beenobserved to act as a substrate for RNaseH, and are believed to act bysterically blocking target RNA nucleocytoplasmic transport, splicing ortranslation. This class includes methylphosphonates (Toulme et al.,1996), morpholino oligonucleotides, peptide nucleic acids (PNA's),certain 2′-O-allyl or 2′-O-alkyl modified oligonucleotides (Bonham,1995), and N3′→P5′ phosphoramidates (Gee, 1998; Ding, 1996).

A test oligomer can be assayed for its RNaseH resistance by forming anRNA:oligomer duplex with the test compound, then incubating the duplexwith RNaseH under a standard assay conditions, as described in Stein etal. After exposure to RNaseH, the presence or absence of intact duplexcan be monitored by gel electrophoresis or mass spectrometry.

A3. In Vivo Uptake

In accordance with another aspect of the invention, there is provided asimple, rapid test for confirming that a given antisense oligomer typeprovides the required characteristics noted above, namely, high T_(m),ability to be actively taken up by the host cells, and substantialresistance to RNaseH. This method is based on the discovery that aproperly designed antisense compound will form a stable heteroduplexwith the complementary portion of the viral RNA target when administeredto a mammalian subject, and the heteroduplex subsequently appears in theurine (or other body fluid). Details of this method are also given inco-owned U.S. patent application Ser. No. 09/736,920, entitled“Non-Invasive Method for Detecting Target RNA” (Non-Invasive Method),the disclosure of which is incorporated herein by reference.

Briefly, a test oligomer containing a backbone to be evaluated, having abase sequence targeted against a known RNA, is injected into a mammaliansubject. The antisense oligomer may be directed against anyintracellular RNA, including a host RNA or the RNA of an infectingvirus. Several hours (typically 8-72) after administration, the urine isassayed for the presence of the antisense-RNA heteroduplex. Ifheteroduplex is detected, the backbone is suitable for use in theantisense oligomers of the present invention.

The test oligomer may be labeled, e.g. by a fluorescent or a radioactivetag, to facilitate subsequent analyses, if it is appropriate for themammalian subject. The assay can be in any suitable solid-phase or fluidformat. Generally, a solid-phase assay involves first binding theheteroduplex analyte to a solid-phase support, e.g., particles or apolymer or test-strip substrate, and detecting the presence/amount ofheteroduplex bound. In a fluid-phase assay, the analyte sample istypically pretreated to remove interfering sample components. If theoligomer is labeled, the presence of the heteroduplex is confirmed bydetecting the label tags. For non-labeled compounds, the heteroduplexmay be detected by immunoassay if in solid phase format or by massspectroscopy or other known methods if in solution or suspension format.

When the antisense oligomer is complementary to a virus-specific regionof the viral genome (such as 5′ end terminal region of the viral RNA, asdescribed above, the method can be used to detect the presence of agiven ssRNA virus, or reduction in the amount of virus during atreatment method.

B. Exemplary Oligomer Backbones

Examples of nonionic linkages that may be used in oligonucleotideanalogs are shown in FIGS. 1A-1G. In these figures, B represents apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide,preferably selected from adenine, cytosine, guanine and uracil. Suitablebackbone structures include carbonate (FIG. 1A, R=O) and carbamate (FIG.1A, R=NH₂) linkages (Mertes and Coats 1969; Gait, Jones et al. 1974);alkyl phosphonate and phosphotriester linkages (FIG. 1B, R=alkyl or—O-alkyl) (Lesnikowski, Jaworska et al. 1990); amide linkages (FIG. 1C)(Blommers, Pieles et al. 1994); sulfone and sulfonamide linkages (FIG.1D, R₁, R₂=CH₂) (Roughten, 1995; McElroy, 1994); and a thioformacetyllinkage (FIG. 1E) (Matteucci, 1990; Cross, 1997). The latter is reportedto have enhanced duplex and triplex stability with respect tophosphorothioate antisense compounds (Cross, 1997). Also reported arethe 3′-methylene-N-methylhydroxyamino compounds of the structure in FIG.1F (Mohan, 1995).

Peptide nucleic acids (PNAs) (FIG. 1G) are analogs of DNA in which thebackbone is structurally homomorphous with a deoxyribose backbone,consisting of N-(2-aminoethyl) glycine units to which pyrimidine orpurine bases are attached. PNAs containing natural pyrimidine and purinebases hybridize to complementary oligonucleotides obeying Watson-Crickbase-pairing rules, and mimic DNA in terms of base pair recognition(Egholm et al., 1993). The backbone of PNAs are formed by peptide bondsrather than phosphodiester bonds, making them well-suited for antisenseapplications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNAduplexes which exhibit greater than normal thermal stability. PNAs arenot recognized by nucleases or proteases.

A preferred oligomer structure employs morpholino-based subunits bearingbase-pairing moieties, joined by uncharged linkages, as described above.Especially preferred is a substantially unchargedphosphorodiamidate-linked morpholino oligomer, such as illustrated inFIGS. 2A-2D. Morpholino oligonucleotides, including antisense oligomers,are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and5,506,337, all of which are expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: theability to be linked in a oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g. adenine,cytosine, guanine or uracil) such that the polymer formed can hybridizewith a complementary-base target nucleic acid, including target RNA,with high T_(m), even with oligomers as short as 10-14 bases; theability of the oligomer to be actively transported into mammalian cells;and the ability of the oligomer:RNA heteroduplex to resist RNAsedegradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the β-morpholino subunit types shown in FIGS. 2A-2D,each linked by an uncharged, phosphorus-containing subunit linkage. FIG.2A shows a phosphorus-containing linkage which forms the five atomrepeating-unit backbone, where the morpholino rings are linked by a1-atom phosphoamide linkage. FIG. 2B shows a linkage which produces a6-atom repeating-unit backbone. In this structure, the atom Y linkingthe 5′ morpholino carbon to the phosphorus group may be sulfur,nitrogen, carbon or, preferably, oxygen. The X moiety pendant from thephosphorus may be fluorine, an alkyl or substituted alkyl, an alkoxy orsubstituted alkoxy, a thioalkoxy or substituted thioalkoxy, orunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures, such as morpholines or piperidines. Alkyl, alkoxy andthioalkoxy preferably include 1-6 carbon atoms. The Z moieties aresulfur or oxygen, and are preferably oxygen.

The linkages shown in FIGS. 2C and 2D are designed for 7-atomunit-length backbones. In Structure 3C, the X moiety is as in Structure3B, and the moiety Y may be methylene, sulfur, or, preferably, oxygen.In Structure 2D, the X and Y moieties are as in Structure 2B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 2B,where X=NH₂ or N(CH₃)₂, Y=O, and Z=O.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages, more preferably up to about 1 per every 10uncharged linkages. Therefore a small number of charged linkages, e.g.charged phosphoramidate or phosphorothioate, may also be incorporatedinto the oligomers.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above. Insome cases, it may be desirable to add additional chemical moieties tothe antisense compound, e.g. to enhance pharmacokinetics or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to a terminus of the oligomer, accordingto standard synthetic methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an antisense oligomer, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

V. Inhibition of Viral Replication

The antisense compounds detailed above are useful in inhibitingreplication of ssRNA viruses of the Flaviviridae, Picornoviridae,Caliciviridae, Togaviridae, Arteriviridae, Coronaviridae, Astroviridaeand Hepeviridae virus families. In one embodiment, such inhibition iseffective in treating infection of a host animal by these viruses.Accordingly, the method comprises, in one embodiment, contacting a cellinfected with the virus with an antisense agent effective to inhibit thereplication of the specific virus. In this embodiment, the antisenseagent is administered to a mammalian subject, e.g., human or domesticanimal, infected with a given virus, in a suitable pharmaceuticalcarrier. It is contemplated that the antisense oligonucleotide arreststhe growth of the RNA virus in the host. The RNA virus may be decreasedin number or eliminated with little or no detrimental effect on thenormal growth or development of the host.

A. Identification of the Infective Agent

The specific virus causing the infection can be determined by methodsknown in the art, e.g. serological or cultural methods, or by methodsemploying the antisense oligomers of the present invention.

Serological identification employs a viral sample or culture isolatedfrom a biological specimen, e.g., stool, urine, cerebrospinal fluid,blood, etc., of the subject. Immunoassay for the detection of virus isgenerally carried out by methods routinely employed by those of skill inthe art, e.g., ELISA or Western blot. In addition, monoclonal antibodiesspecific to particular viral strains or species are often commerciallyavailable.

Culture methods may be used to isolate and identify particular types ofvirus, by employing techniques including, but not limited to, comparingcharacteristics such as rates of growth and morphology under variousculture conditions.

Another method for identifying the viral infective agent in an infectedsubject employs one or more antisense oligomers targeting broad familiesand/or genera of viruses, e.g., Picornaviridae, Caliciviridae,Togaviridae and Flaviviridae. Sequences targeting any characteristicviral RNA can be used. The desired target sequences are preferably (i)common to broad virus families/genera, and (ii) not found in humans.Characteristic nucleic acid sequences for a large number of infectiousviruses are available in public databases, and may serve as the basisfor the design of specific oligomers.

For each plurality of oligomers, the following steps are carried out:(a) the oligomer(s) are administered to the subject; (b) at a selectedtime after said administering, a body fluid sample is obtained from thesubject; and (c) the sample is assayed for the presence of anuclease-resistant heteroduplex comprising the antisense oligomer and acomplementary portion of the viral genome. Steps (a)-(c) are carried forat least one such oligomer, or as many as is necessary to identify thevirus or family of viruses. Oligomers can be administered and assayedsequentially or, more conveniently, concurrently. The virus isidentified based on the presence (or absence) of a heteroduplexcomprising the antisense oligomer and a complementary portion of theviral genome of the given known virus or family of viruses.

Preferably, a first group of oligomers, targeting broad families, isutilized first, followed by selected oligomers complementary to specificgenera and/or species and/or strains within the broad family/genusthereby identified. This second group of oligomers includes targetingsequences directed to specific genera and/or species and/or strainswithin a broad family/genus. Several different second oligomercollections, i.e. one for each broad virus family/genus tested in thefirst stage, are generally provided. Sequences are selected which are(i) specific for the individual genus/species/strains being tested and(ii) not found in humans.

B. Administration of the Antisense Oligomer

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. In accordance with the invention,routes of antisense oligomer delivery include, but are not limited to,various systemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of an antisense oligomer in the treatment of a viralinfection of the skin is topical delivery, while delivery of anantisense oligomer for the treatment of a viral respiratory infection isby inhalation. The oligomer may also be delivered directly to the siteof viral infection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically accepted carriers employed bythose of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12): 1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., ANTISENSE OLIGONUCLEOTIDES: A NEWTHERAPEUTIC PRINCIPLE, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic viral infection. Thecondition of a patient may also dictate prophylactic administration ofan antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc, and the treatment iseither prophylactic or therapeutic. The invention also includes alivestock and poultry food composition containing a food grainsupplemented with a subtherapeutic amount of an antiviral antisensecompound of the type described above. Also contemplated is, in a methodof feeding livestock and poultry with a food grain supplemented withsubtherapeutic levels of an antiviral, an improvement in which the foodgrain is supplemented with a subtherapeutic amount of an antiviraloligonucleotide composition as described above.

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer. Typically, one or more doses of antisense oligomerare administered, generally at regular intervals, for a period of aboutone to two weeks. Preferred doses for oral administration are from about1-100 mg oligomer per 70 kg. In some cases, doses of greater than 100 mgoligomer/patient may be necessary. For i.v. administration, preferreddoses are from about 0.5 mg to 100 mg oligomer per 70 kg. The antisenseoligomer may be administered at regular intervals for a short timeperiod, e.g., daily for two weeks or less. However, in some cases theoligomer is administered intermittently over a longer period of time.Administration may be followed by, or concurrent with, administration ofan antibiotic or other therapeutic treatment. The treatment regimen maybe adjusted (dose, frequency, route, etc.) as indicated, based on theresults of immunoassays, other biochemical tests and physiologicalexamination of the subject under treatment.

C. Monitoring of Treatment

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of viral infection under treatment,and corresponding adjustments in the dose or treatment regimen, in orderto achieve an optimal therapeutic outcome. Treatment may be monitored,e.g., by general indicators of infection, such as complete blood count(CBC), nucleic acid detection methods, immunodiagnostic tests, viralculture, or detection of heteroduplex.

The efficacy of an in vivo administered antisense oligomer of theinvention in inhibiting or eliminating the growth of one or more typesof RNA virus may be determined from biological samples (tissue, blood,urine etc.) taken from a subject prior to, during and subsequent toadministration of the antisense oligomer. Assays of such samples include(1) monitoring the presence or absence of heteroduplex formation withtarget and non-target sequences, using procedures known to those skilledin the art, e.g., an electrophoretic gel mobility assay; (2) monitoringthe amount of viral protein production, as determined by standardtechniques such as ELISA or Western blotting, or (3) measuring theeffect on viral titer, e.g. by the method of Spearman-Karber. (See, forexample, Pari, G. S. et al., Antimicrob. Agents and Chemotherapy 39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents andChemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed) in: Manual ofStandard Methods for Veterinary Microbiology, pp. 60-93, 1978).

A preferred method of monitoring the efficacy of the antisense oligomertreatment is by detection of the antisense-RNA heteroduplex. At selectedtime(s) after antisense oligomer administration, a body fluid iscollected for detecting the presence and/or measuring the level ofheteroduplex species in the sample. Typically, the body fluid sample iscollected 3-24 hours after administration, preferably about 6-24 hoursafter administering. As indicated above, the body fluid sample may beurine, saliva, plasma, blood, spinal fluid, or other liquid sample ofbiological origin, and may include cells or cell fragments suspendedtherein, or the liquid medium and its solutes. The amount of samplecollected is typically in the 0.1 to 10 ml range, preferably about 1 mlor less.

The sample may be treated to remove unwanted components and/or to treatthe heteroduplex species in the sample to remove unwanted ssRNA overhangregions, e.g. by treatment with RNase. It is, of course, particularlyimportant to remove overhang where heteroduplex detection relies on sizeseparation, e.g., electrophoresis of mass spectroscopy.

A variety of methods are available for removing unwanted components fromthe sample. For example, since the heteroduplex has a net negativecharge, electrophoretic or ion exchange techniques can be used toseparate the heteroduplex from neutral or positively charged material.The sample may also be contacted with a solid support having asurface-bound antibody or other agent specifically able to bind theheteroduplex. After washing the support to remove unbound material, theheteroduplex can be released in substantially purified form for furtheranalysis, e.g., by electrophoresis, mass spectroscopy or immunoassay.

VI. Heteroduplex Complex

In another aspect, the invention includes a heteroduplex complex formedbetween:

(a) a region within the 5′-terminal 40 bases of the positive strand RNAof an RNA virus having a single-stranded, positive-sense RNA genome andselected from one of the Flaviviridae, Picornoviridae, Caliciviridae,Togaviridae, Arteriviridae, Coronaviridae, Astroviridae or Hepeviridaefamilies, which region is capable of forming internal stem-loopsecondary structure, and

(b) an oligonucleotide analog compound characterized by:

(i) a nuclease-resistant backbone,

(ii) capable of uptake by mammalian host cells,

(iii) containing between 12-40 nucleotide bases,

(iv) having a targeting sequence of at least 12 subunits that iscomplementary to a region associated with such stem-loop secondarystructure within the 5′-terminal end 40 bases of the positive-sense RNAstrand of the virus,

where said heteroduplex complex has a Tm of dissociation of at least 45°C. and disruption of such stem-loop secondary structure.

An exemplary compound is composed of morpholino subunits linked byuncharged, phosphorus-containing intersubunit linkages, joining amorpholino nitrogen of one subunit to a 5′ exocyclic carbon of anadjacent subunit. The compound may have phosphorodiamidate linkages,such as in the structure

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In apreferred compound, X=NR₂, where each R is independently hydrogen ormethyl. The compound may be the oligonucleotide analog alone or aconjugate of the analog and an arginine-rich polypeptide capable ofenhancing the uptake of the compound into host cells.

In one embodiment, the compound is effective, when administered to thehost cells, to form a heteroduplex structure (i) composed of thepositive sense strand of the virus and the oligonucleotide compound, and(ii) characterized by a Tm of dissociation of at least 45° C. anddisruption of such stem-loop secondary structure.

EXAMPLES

The following examples illustrate but are not intended in any way tolimit the invention.

Materials and Methods

Standard recombinant DNA techniques were employed in all constructions,as described in Ausubel, F M et al., in CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley and Sons, Inc., Media, Pa., 1992 and Sambrook, J. etal., in MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vol. 2, 1989).

All peptides were custom synthesized by Global Peptide Services (Ft.Collins, Colo.) or at AVI BioPharma (Corvallis, Oreg.) and purifiedto >90% purity (see Example 2 below). PMOs were synthesized at AVIBioPharma in accordance with known methods, as described, for example,in (Summerton and Weller 1997) and U.S. Pat. No. 5,185,444.

PMO oligomers were conjugated at the 5′ end with an arginine-richpeptide (R₉F₂C-5′-PMO) to enhance cellular uptake as described (U.S.Patent Application 60/466,703 and (Moulton, Nelson et al. 2004).

Example 1 Antisense Inhibition of Flaviviridae (Yellow Fever Virus) InVitro

Although an effective vaccine for yellow fever virus (YFV) has beenavailable for many years, this virus continues to be a leading cause ofhemorrhagic fever with mortality rates as high as 50%. Worldwide, thereare 200,000 estimated cases of yellow fever (with 30,000 deaths)annually. Small numbers of imported cases also occur in countries freeof yellow fever (WHO, Fact Sheet 100, 2001).

A PMO antisense oligomer targeted to the 5′ positive strand terminus ofYFV (SEQ ID NO:49) was evaluated in a 4-concentration test. The standardCPE test used an 18 h monolayer (80-100% confluent) of Vero cells,medium was drained and each of the concentrations of PMO or scramblecontrol sequence was added, followed within 15 min by virus or virusdiluent. Two wells are used for each concentration of compound for bothantiviral and cytotoxicity testing. The plate was sealed and incubatedthe standard time period required to induce near-maximal viral CPE. Theplate was then stained with neutral red by the method described belowand the percentage of uptake indicating viable cells read on amicroplate autoreader at dual wavelengths of 405 and 540 nm, with thedifference taken to eliminate background. An approximatedvirus-inhibitory concentration, 50% endpoint (EC50) and cell-inhibitoryconcentration, 50% endpoint (IC50) was determined from which a generalselectivity index (S.I.) was calculated: S.I.=(IC50)/(EC50). An SI of 3or greater indicates significant antiviral activity. The PMO targetingthe 5′ positive-strand terminal region (SEQ ID NO:49) produced an SI of21 in this assay.

Example 2 Antisense Inhibition of Flaviviridae (Dengue Virus Serotypes1-4) In Vitro

Dengue Fever/Dengue Hemorrhagic Fever (DF/DHF) has become a major globalhealth problem over the past 20 years. Geographic distribution of thedengue virus (DEN), its mosquito vectors and the disease burden itcauses continue to increase. The World Health Organization estimatesthat there are 50-100 million new infections yearly. DF/DHF is now aleading cause of hospitalization and death among children in southernAsia, and its incidence is sharply rising in the Americas. There iscurrently no vaccine or effective therapeutic. One requirement of asuccessful vaccine or therapeutic is that it be effective against all 4human serotypes of DEN. The purpose of this study was to evaluate theefficacy and specificity of PMO that target the 5′ positive-strandterminal stem loop at inhibiting the replication of four serotypes ofDEN in Vero cells in culture. The PMO was designed to target thesequence element in the positive-strand DEN2 RNA that may be importantin viral transcription and/or translation (Markoff 2003). The PMO inthis study were conjugated to an arginine-rich peptide in order tofacilitate entry into Vero E6 cells (Moulton, Nelson et al. 2004;Neuman, Stein et al. 2004).

A PMO, 5′SL, (SEQ ID NO:51) designed to hybridize to the 5′ positivestrand terminal region of Dengue 2 virus (DEN2), were evaluated fortheir ability to inhibit Dengue virus replication in mammalian cellculture. The PMOs were conjugated to a short arginine-rich peptide(R₉F₂C-5′-PMO) to facilitate their entry into cells in culture. Vero E6cells were incubated with the PMO agents, inoculated with DEN serotypes1-4 (DEN1, DEN2, DEN3, DEN4, respectively), and viral titer determinedby plaque-assay 5-8 days later. The compound targeting the 5′ positivestrand terminus (5′SL) reduced the titer of DEN2 by over 4 orders ofmagnitude, compared to controls, in a dose-dependent andsequence-specific manner over a 4 day period as shown in FIG. 5A. Ten μMsolutions of the 5′SL PMO reduced the titer of all four Dengue serotypesby over two to four orders of magnitude, in some cases below detectablelimits as shown in FIGS. 5A-5B. The 5′SL PMO was less effective againstDEN4 (two log reduction) than it was against DEN1, DEN2 and DEN3 (fourlog reductions) due to a two base pair mismatch between the 5′SL PMO andit's target sequence in DEN4. The effective anti-DEN compounds did notalter the titer of West Nile Virus (WNV) grown in Vero E6 cells. Thisdata indicates that the 5′SL PMO compound is a potential DEN 1-4therapeutic.

Example 3 Antisense Inhibition of Coronaviridae (Porcine Reproductiveand Respiratory Syndrome Virus PRRSV) In Vitro

Porcine reproductive and respiratory syndrome (PRRS) is a contagiousviral disease that is characterized by reproductive failure in sows andrespiratory disease in young pigs. The causative agent, PRRSV, is asingle-stranded RNA virus with genome organization similar to that ofother members of the Coronaviridae. PRRS causes heavy economic losses tothe swine industry though a vaccine has been widely used for years.Specific anti-PRRSV drugs are urgently needed as one of the integratedstrategies to prevent and control PRRSV infection. A PMO (PRRSV-1a, SEQID NO: 109) that targets the 5′ positive strand terminal region of PRRSVwas tested for its ability to inhibit viral replication as describedbelow.

The first test was designed to determine whether the PRRSV-1a PMO couldinhibit the development of virus-induced, cell pathogenic effect (CPE).ATCC CRL11171 cells were used for this experiment as previouslyreported. The CRL11171 monolayer cells were treated with the PRRSV-1aPMO (SEQ ID NO:109) in DMEM for 4 h at 37° C. The PMOs were removed fromthe cells and inoculated with PRRSV strain VR2385 at a multiplicity ofinfection (MOI) of one. The cells were cultured and observed daily forCPE development. A blank control and the control PMO (DSscr, a scramblesequence PMO) were included as negative controls. The cell culturemedium was also collected and titrated in CRL11171 cells to determinePRRSV titer.

The PRRSV-1a PMO targeting the 5′ positive-strand end-terminus of theuntranslated region (UTR), was found to be effective in inhibiting PRRSVreplication (FIG. 6). The cells treated with PRRSV-1a PMO at 16 μM hadmuch less cell pathogenic effect (CPE) development than controls (FIG.7). CPE is clearly visible after PRRSV infection (positive), whileuninfected control cells remain an intact monolayer (blank). PMOPRRSV-1a reduced CPE development, while other PMOs including control PMOdid not have much effect in blocking CPE. The cells and medium wereharvested for titration of PRRSV yield. Tissue culture infectious dose(TCID50) was calculated based on CPE development of different dilutions.PRRSV-1a reduced virus yield by more than 90% (not shown).

The inhibition of PRRSV replication by the PRRSV-1a PMO was also shownto be dose-dependent. Using the PMO-treatment and virus cultureconditions described above, three different concentrations of PRRSV-1aand DSscr control PMO (4, 8 and 16 mM) were tested on PRRSV-infectedCRRL11171 cells for the ability to inhibit viral replication as measuredby viral titer. As shown in FIG. 6, the PRRSV-1a PMO inhibits PRRSVreplication in a dose-dependent manner.

Example 4 Antisense Inhibition of Tick Borne Encephalitis Virus

This example describes a study that was devised to test the antiviralactivity of antisense PMO compounds of the present invention against twoflaviviruses; Tick Borne Encephalitis virus (TBE) and West Nile virus(WNV). Two PMO oligomers were evaluated for antiviral activity; TBE5′SL, SEQ ID NO:57 and; a scramble control sequence DSscr(5′-AGTCTCGACTTGCTACCTCA-3′ SEQ ID NO:133). Both PMO oligomers wereconjugated at the 5′ end with an arginine-rich peptide (R₉F₂C-5′-PMO) toenhance cellular uptake as described (U.S. Patent Application 60/466,703and (Moulton, Nelson et al. 2004). The WNV infection provided a negativecontrol infection as there is no homology between WNV and the TBE 5′SLtargeting PMO. This control indicates the level of non-specific viralsuppression of each of the PMOs. The PMO compounds were prepared toprovide a 2 mM stock solution, which were then titrated against astandard dose of virus on tissue culture cells. Cells were infected witha multiplicity of infection (MOI) of 1 and the virus yield was assessedin samples of supernatant medium taken at 18 hours post infection.

The two virus strains used in this example:

-   -   1) TC 401 West Nile 99-34940-31A (New York strain) Passage 2    -   2) TC 339 Tick Borne Encephalitis virus (Hypr strain) Passage 49

Four T175 tissue culture flasks (NUNC) of SW 13 cells (human caucasianadrenal cortex adenocarcinoma cell line ECAAC 87031801 grown in RPMI1640 medium plus 5% FBS) at passage 130 were washed twice withtrypsin-EDTA (1×) and incubated for 2-3 minutes at 37° C. The cells wereresuspended in 11.5 ml growth medium per flask and pooled. A cell countwas performed on the pooled cell suspension and the result was 1.74×10⁶cells/ml with 99% viability. Six mls of the cell suspension was used toseed four T175 flasks and 40 ml of the cell suspension was diluted to270 ml. This was dispensed in 3 ml aliquots per well in 15 six-wellplates. The plates were incubated overnight to form confluent cellmonolayers.

Each of the PMO compounds was diluted to 25, 20, 15, 10 and 5 μM in 4 mlserum-free RPMI 1640 medium. The medium was removed from the wells oftwo six-well plates. 2 ml of the appropriate compound dilution wasdispensed in all wells of a plate and this was repeated on separateplates for both PMO compounds. The plates were incubated at 37° C. for 5hours. The two viruses were removed from the −70° C. freezer and thawedrapidly. Each virus was diluted to 2×10⁶ pfu/ml to produce 42 mlserum-free medium. The six-well plates were removed from the incubatorand the pre-treatment medium aspirated from all the wells. 1 ml ofmedium was added to each well of the control plate (no compound). Eachset of plates received 1 ml/well of either TBE or WN diluted to 2×10⁶pfu/ml. The plates were incubated at room temperature for 1 hour and themedium was then removed and replaced with 2 ml RPMI 1640 plus 1% FBSplus the same concentration of test compound as used to pre-treat thecells. The plates were incubated at 37° C. for 18 hours.

To prepare 24 well plates for determining virus titers, eight T175tissue culture flasks (NUNC) of SW 13 cells at passage 131 were washedtwice with trypsin-EDTA (1×) and incubated for 2-3 minutes at 37° C. Thecells were resuspended in 11.5 ml growth medium per flask and pooled. Acell count was performed on the pooled cell suspension and the resultwas 1.7×10⁶ cells/ml with 99% viability. 80 ml of the cell suspensionwas diluted to 680 ml. These cells were dispensed as 1 ml per wellaliquots in eight 24-well plates. The plates were incubated overnight toform confluent monolayers.

At 18 hours post-infection the supernatant media from the PMO-treated,virus-infected six-well plates were harvested from each individualwells. Thirty microliters of each harvest was placed in a single cup ofa 96-well plate with 270 microliters serum-free medium. The remainder ofthe sample was placed in cryotube and stored at −70° C. The medium wasremoved from the 24-well plates and 250 μl of the titration dilutionswere transferred from the 96-well plates to the 24 well plates whichwere incubated at 37° C. for one hour. One ml agarose overlay medium wasadded to each well and after allowing the agarose to set at roomtemperature the plates were incubated at 37° C. for 5 days. After 5 daysthe plates were removed from the incubator, 1 ml 10% Formol saline wasadded to each well and the plates were left at room temperature for 3hours. The plates were washed under running water to remove the agarosemedium and left to drain inverted while the remaining plates werewashed. Each well then received 1 ml of 0.1% Naphthalene black stain andthe plates were left for 30 minutes before the stain was removed and theplates washed under running water. They were then left to dry (inverted)for 3 hours. Viral plaques were counted to determine the titer.

FIG. 8 shows the viral titer obtained from the PMO-treated infections asa percentage of untreated control, with virus-infected cells infectedwith either TBEV or WNV and treated with either the TBEV antisensecompound where the PMO compound is either 5′SL (SEQ ID NO:57) or controlPMO (DSscr, a scrambled base sequence). As seen from a comparison of theviral titers in FIG. 8, significant there is a reduction in viral titrein all cells (treated and control) with increasing concentrations ofcompound, thought to be due to a cell-toxicity effect of the attachedarginine-rich peptide present in both antisense and control compounds.However, at compound concentrations of 5 μM and above, there is seen asequence-specific increase in TBEV inhibition, both relative to WNV(FIG. 8), and relative to the DSscr scrambled control sequence.

Example 5 Effect of PMO on West Nile Virus (WNV) Infection in Mice

PMOs are uncharged, water-soluble, nuclease-resistant antisense agentsthat are typically synthesized to a length of about 20 subunits andcontain purine and pyrimidine bases attached to a backbone composed ofmorpholine rings joined by phosphorodiamidate intersubunit linkages. Inexperiments in support of the invention, it was shown that conjugationof an Arg-rich peptide (designated as P007; SEQ ID NO:122) to the 5′-endof the PMOs greatly facilitates the delivery of the PMO into culturedcells. P007-PMOs targeting different regions of the viral genome haveinhibited WNV virus infection to various degrees. Among them, PMOstargeting the 5′-terminal 20 nucleotides (5′End; SEQ ID NO:47) showedpotent antiviral activity.

Experiments were conducted in mice to extend the observations to in vivoconditions. Female BALB/c mice were used. The mice were obtained fromSimonsen Laboratories (Gilroy, Calif.). At the time that the experimentwas started, the animals had been in the animal facility for one weekand they were greater than 6 weeks of age. The mice weighed 12.3 to 19.8g with an average of 16.1 g. Experiments were conducted in the BSL-3animal suite at Utah State University Laboratory Animal Research Center(LARC). Two PMO compounds were used: 1) a PMO targeting the 5′ terminus(NG040006; SEQ ID NO:47) and; 2) the same PMO conjugated at its 5′ endwith the P007 arginine-rich peptide (SEQ ID NO: 122) and named NG040005.NG040007 is an unconjugated, scramble control PMO. Ampligen™ was used asa positive control, antiviral compound and was obtained from William M.Mitchell (School of Medical Pathology, Vanderbilt University, Nashville,Tenn. 37240). Since ampligen is an RNA-like molecule, care was used toprevent contamination with RNase by using RNase-free materials andDEPC-treated water.

Ten animals were randomly assigned to each treatment group, except forthe placebo group 11, which had 20 animals. Intraperitoneal treatmentswere initiated 24 hours before subcutaneous WNV challenge. PMOtreatments continued qd, −4 hours before viral challenge, 1, 2, 3, 4, 5and 5 days post-viral injection (dpi). Ampligen was treated i.p., qd,−1, 1, 3, and 5 dpi. Dosages and treatment groups are indicated in thetable below along with Survival and mean day to death (MDD). NG040005and ampligen increased the MDD of WNV-infected mice as compared to theplacebo control (Table 4).

TABLE 4 Effect of PMOs on West Nile virus infection in mice Animals:Female BALB/c mice, >6 wk old Virus: West Nile virus, NY crow brainhomogenate, 10^(6.3) infectious units, s.c. injection Drug diluent:saline Treatment schedule: qd, −1 d, −4 h, 1, 2, 3, 4, 5, 6 d TreatmentRoute: i.p. Duration of experiment: 21 days % survival (alive/ MDD^(a) ±Survival Drug Dose Schedule total) SD analysis^(b) NG040005 250 qd, −1d, 50% 14.0 ±  P = 0.20 μg/inj −4 h, 1, 2, (5/10) 3.8*** 3, 4, 5, 6 dNG040006 750 qd, −1 d, 40% 8.2 ±  P = 0.95 μg/inj −4 h, 1, 2, (4/10) 1.03, 4, 5, 6 d ampligen 14 qd, −1 d, 80% 13.5 ± P ≧ 0.01** mg/kg 1, 3, 5 d(8/10) 4.9 placebo — qd, −1 d, 35% 8.6 ± — −4 h, 1, 2, (7/20) 1.3 3, 4,5, 6 d ^(a)Mean day to death of mice dying prior to day 21. Student'st-test was used for analysis. ^(b)Log-rank survival analysis. Toxicitycontrols were not run in this first experiment because of limitedamounts of compounds. *P ≦ 0.05, **P ≦ 0.01, ***P ≦ 0.001 compared toplacebo.

TABLE 5 Sequence Listing Table SEQ ID NO Sequence, 5′ to 3′ 1GNNGATGTTCGCGTCGGTGAGCGGAGAGGAAACAGATTTC 2AGAAGTTTATCTGTGTGAACTTCTTGGCTTAGTATCGTTG 3AGACGTTCATCTGCGTGAGCTTCCGATCTCAGTATTGTTT 4AGTAGTTCGCCTGTGTGAGCTGACAAACTTAGTAGTGTTT 5AGTAAATCCTGTGTGCTAATTGAGGTGCATTGGTCTGCAA 6AGTTGTTAGTCTACGTGGACCGACAAAGACAGATTCTTTG 7GCCAGCCCCCTGATGGGGGCGACACTCCACCATGAATCAC 8AGATTTTCTTGCACGTGCATGCGTTTGCTTCGGACAGCAT 9AGATTTTCTTGCACGTGCGTGCGCTTGCTTCAGACAGCAA 10AGATTTTCTTGCACGTGTGTGCGGGTGCTTTAGTCAGTGT 11TTAAAACAGCTCTGGGGTTGTACCCACCCCAGAGGCCCAC 12TTAAAACAGCCTGTGGGTTGTACCCACCCACAGGGCCCAC 13TTAAAACAGCCTGTGGGTTGTTCCCACCCACAGGCCCATT 14TTAAAACAGCTCTGGGGTTGCTCCCACCCCAGAGGCCCAC 15TTAAAACAGCTCTGGGGTTGTTCCCACCCCAGAGGCCCAC 16GAGTGTTCCCACCCAACAGGCCCACTGGGTGTTGTACTCT 17TTAAAACAGCCTGGGGGTTGTACCCACCCCTGGGGCCCAC 18TTAAAACTGGGAGTGGGTTGTTCCCACTCACTCCACCCAT 19TTAAAACAGCGGATGGGTATCCCACCATTCGACCCATTGG 20TTGAAAGGGGGCGCTAGGGTTTCACCCCTAGCATGCCAAC 21TTCAAGAGGGGTCTCCGGGAATTTCCGGAGTCCCTCTTGG 22GTAAAAGAAATTTGAGACAATGTCTCAAACTCTGAGCTTC 23GTTAATGAGAAATGGCTTCTGCCATCGCTCTCTCGAGCTC 24GTGATCGTGATGGCTAATTGCCGTCCGTTGCCTATTGGGC 25GTGATTTAATTATAGAGAGATAGTGACTTTCACTTTTCTT 26GTGAATGATGATGGCGTCAAAAGACGTCGTTCCTACTGCT 27GCCATGGAGGCCCATCAGTTTATTAAGGCTCCTGGCATCA 28ATGGAAGCTATCGGACCTCGCTTAGGACTCCCATTCCCAT 29ATATTAGGTTTTTACCTACCCAGGAAAAGCCAACCAACCT 30ACTTAAAAAGATTTTCTATCTACGGATAGTTAGCTCTTTT 31ACTTTTAAAGTAAAGTGAGTGTAGCGTGGCTATATCTCTT 32GATTGCGAGCGATTTGCGTGCGTGCATCCCGCTTCACTGA 33ACTTAAGTACCTTATCTATCTACAGATAGAAAAGTTGCTT 34TATAAGAGTGATTGGCGTCCGTACGTACCCTCTCAACTCT 35ATGACGTATAGGTGTTGGCTCTATGCCTTGGCATTTGTAT 36GCTCGAAGTGTGTATGGTGCCATATACGGCTCACCACCAT 37CCAAGAGGGGGGTGGTGATTGGCCTTTGGCTTATCAGTGT 38ATAGGGTACGGTGTAGAGGCAACCACCCTATTTCCACCTA 39ACCCTACAAACTAATCGATCCAATATGGAAAGAATTCACG 40ATGGGCGGCGCAAGAGAGAAGCCCAAACCAATTACCTACC 41 ACCGACGCGAACATCNNC 42TCCTCTCCGCTCACCGACGC 43 TCACACAGATAAACTTCT 44 AAGCCAAGAAGTTCACACAG 45TCACGCAGATGAACGTCT 46 GAGATCGGAAGCTCACGCAG 47 GCTCACACAGGCGAACTACT 48TAAGTTTGTCAGCTCACACAG 49 CAATTAGCACACAGGATTTACT 50 TTGCAGACCAATGCACCTCA51 GTCCACGTAGACTAACAACT 52 GTCTTTGTCGGTCCACGTAG 53 CCCATCAGGGGGCTGGC 54TGGAGTGTCGCCCCCATCAG 55 ATGCACGTGCAAGAAAATCT 56 ATGCTGTCCGAAGCAAACGC 57CACGCACGTGCAAGAAAATCT 58 TGAAGCAAGCGCACGCACGT 59 ACACACGTGCAAGAAAATCT 60ACACTGACTAAAGCACCCGC 61 GGTACAACCCCAGAGCTGTTTTAA 62 GTGGGCCTCTGGGGTGGGTA63 CAACCCACAGGCTGTTTTAA 64 GTGGGCCCTGTGGGTGGGTA 65 CAACCCACAGGCTGTTTTAA66 AATGGGCCTGTGGGTGGGAA 67 CAACCCCAGAGCTGTTTTAA 68 GTGGGCCTCTGGGGTGGGAG69 CAACCCCAGAGCTGTTTTAA 70 GTGGGCCTCTGGGGTGGGAA 71 CCTGTTGGGTGGGAACACTC72 AGAGTACAACACCCAGTGGG 73 CAACCCCCAGGCTGTTTTAA 74 GTGGGCCCCAGGGGTGGGTA75 CAACCCACTCCCAGTTTTAA 76 ATGGGTGGAGTGAGTGGGAA 77 ATACCCATCCGCTGTTTTAA78 CCAATGGGTCGAATGGTGGG 79 AACCCTAGCGCCCCCTTTCAA 80 GTTGGCATGCTAGGGGTGAA81 TCCCGGAGACCCCTCTTGAA 82 CCAAGAGGGACTCCGGAAAT 83 TTGTCTCAAATTTCTTTTAC84 GAAGCTCAGAGTTTGAGACA 85 AGAAGCCATTTCTCATTAAC 86 GAGCTCGAGAGAGCGATGGC87 CAATTAGCCATCACGATCAC 88 GGCAACGGACGGCAATTAGC 89 TCTCTCTATAATTAAATCAC90 AAAGTCACTATCTCTCTATA 91 TTGACGCCATCATCATTCAC 92 AGCAGTAGGAACGACGTCTT93 AACTGATGGGCCTCCATGGC 94 TGATGCCAGGAGCCTTAATA 95 CGAGGTCCGATAGCTTCCAT96 ATGGGAATGGGAGTCCTAAG 97 GGTAGGTAAAAACCTAATAT 98 AGGTTGGTTGGCTTTTCCTG99 GATAGAAAATCTTTTTAAGT 100 AAAAGAGCTAACTATCCGTA 101ACTCACTTTACTTTAAAAGT 102 GCCACGCTACACTCACTTTA 103 CACGCAAATCGCTCGCAATC104 TCAGTGAAGCGGGATGCACG 105 GATAGATAAGGTACTTAAGT 106AAGCAACTTTTCTATCTGTA 107 CGGACGCCAATCACTCTTATA 108GAGTTGAGAGGGTACGTACGGA 109 CATAGAGCCAACACCTATACG 110ATACAAATGCCAAGGCATAG 111 GCACCATACACACTTCGAGC 112 ATGGTGGTGAGCCGTATATG113 AATCACCACCCCCCTCTTGG 114 GCCAAAGGCCAATCACCACC 115GCCTCTACACCGTACCCTAT 116 TAGGTGGAAATAGGGTGGTT 117 GATCGATTAGTTTGTAGGGT118 CGTGAATTCTTTCCATATTG 119 TTCTCTCTTGCGCCGCCCAT 120GGTAGGTAATTGGTTTGGGC Peptide SEQ Sequences ID Name (NH₂ to COOH) NO P003RRRRRRRRRFFC 121 P007 (RAhxR)₄AhxβAla 122 P008 (RAhx)₈βAla 123 RX4(RAhx)₄βAla 124 RXR2 (RAhxR)₂AhxβAla 125 RXR3 (RAhxR)₃AhxβAla 126 SEQ IDNO Sequence, 5′ to 3′ 127 ACAGGCGAACTACT 128 GTCAGCTCACAC 129GCTCACACAGGCGA 130 GCACACAGGATTTACT 131 GTCCAATGCACCTC 132CAATGCACCTCAATTAGC 133 AGTCTCGACTTGCTACCTCA

It is claimed:
 1. A method of inhibiting replication of a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising: administering to mammalian host cells a virus-inhibitory amount of an antisense oligonucleotide analog characterized by (i) a nuclease-resistant backbone, (ii) being capable of uptake by mammalian host cells, (iii) containing up to 25 nucleotide bases, (iv) being composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, and (v) having a targeting sequence that comprises the sequence set forth in SEQ ID NO:47, 49, 51, or 55, wherein the antisense oligonucleotide analog disrupts a stem-loop structure in the 5′-terminal region of the RNA virus and inhibits virus production.
 2. The method of claim 1, wherein the heteroduplex has a Tm of dissociation of at least 45° C.
 3. The method of claim 1, wherein the intersubunit linkages are uncharged.
 4. The method of claim 1, wherein the morpholino subunits are joined by intersubunit linkages, in accordance with the structure:

where Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide; and X is alkyl, alkoxy, thioalkoxy; or —NR₂, where each R is independently hydrogen or lower alkyl.
 5. The method of claim 4, wherein X is —NR₂, wherein R is independently hydrogen or methyl.
 6. The method of claim 1, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.
 7. The method of claim 6, wherein the arginine-rich polypeptide has the sequence of SEQ ID NO:
 122. 8. The method of claim 1, wherein the administration is to a mammalian subject in a therapeutically effective amount to treat a Flaviviridae virus infection.
 9. The method of claim 8, wherein the Flaviviridae virus is Dengue virus.
 10. The method of claim 1, wherein the administration is by intravenous or oral administration.
 11. The method of claim 1, wherein the antisense oligonucleotide analog administered is contained in a cocktail of antisense oligonucleotides directed against two or more Flaviviridae positive-strand RNA viruses.
 12. A method of treating an infection by a Flaviviridae positive strand RNA virus selected from yellow fever virus, Dengue virus, tick born encephalitis virus, and West Nile virus, comprising: administering to a subject a therapeutically effective amount of an antisense morpholino oligonucleotide, wherein the morpholino oligonucleotide has (i) a targeting sequence that comprises the sequence set forth in SEQ ID NO: 47, 49, 51, or 55, (ii) a subunit length of up to 25 subunits, wherein morpholino subunits of the oligomer are joined by intersubunit linkages in accordance with the structure:

wherein Y₁=O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide; and X is —NR₂, where each R is independently hydrogen or methyl; and wherein the antisense morpholino oligomer forms a heteroduplex with the target region to disrupt the stem loop structure and inhibit Flaviviridae virus production.
 13. The method of claim 12, wherein the Flaviviridae virus is a Dengue virus and the targeting sequence comprises SEQ ID NO:51.
 14. The method of claim 12, wherein the Flaviviridae is a yellow fever virus and the targeting sequence comprises SEQ ID NO:49.
 15. The method of claim 12, wherein the Flaviviridae is a tick borne encephalitis virus and the targeting sequence comprises SEQ ID NO:55.
 16. The method of claim 12, wherein the Flaviviridae is a West Nile virus and the targeting sequence comprises SEQ ID NO:47.
 17. The method of claim 13, wherein the antisense oligonucleotide has a subunit length of about 22 subunits.
 18. The method of claim 17, wherein the antisense oligonucleotide is 22 subunits in length and is 100% complementary to SEQ ID NO:6.
 19. The method of claim 14, wherein the antisense oligonucleotide is attached to an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells.
 20. The method of claim 19, wherein the arginine-rich polypeptide has the sequence set forth in SEQ ID NO:
 122. 